Cancer Medications

thank you, dennis for that kind introduction.yes, i've been very lucky that i have very, very good mentors in my life, and hopefullyi will get to repay that favor. i'm going to tell you a little bit about marine naturalproducts as potential treatments for pancreatic cancer. at first, we're going to start withtelling you what a natural product is. these are compounds that get made by organisms thatare not essential for the organism to survive. because they take energy to be made, we assumethat they confer some sort of advantage to the organism by making it, so that is thereason they make it. they also are called secondary metabolites, so whenever you hearnatural product or secondary metabolite, that's what we're talking about.why explore the oceans for medicines? a lot

of the drugs, over 62 percent of the drugsthat we currently use, can be traced back to a natural product. some examples are aspirin,morphine, penicillin, and taxol, and taxol is the most used drug against cancer rightnow. of course, all of these are of terrestrial origin, but the oceans cover over 70 percentof the earth's surface, so it makes sense that if we found this many from terrestrialsources, we're going to find more from the ocean. there are more than 200,000 describedspecies of plants and animals in the sea, and there are many left to be discovered.of this, the small percentage that have been studied, have already yielded 10,000 novelchemicals, but only a fraction of these have been tested for pharmaceutical potential.as you can see, this is an untapped source

of potential new drugs.some examples of drugs that came from the sea that are used against cancer are yondelis,or ecteinascidin 743. this is an anti-tumor drug, and this is the structure and this iswhat the sea squirt that it comes from looks like. it's an anti-tumor drug from ecteinascidiaturbinata, or a sea squirt or tunicate that is found in the caribbean and mediterraneanseas. it is also off the coast of fort pierce; you can get it right here. actually, dr. amywright helped find the structure of this compound, and that's very important because withoutthat, you cannot get the synthesis and make it in the lab. so it is very essential thatwe always know the structure of the compounds. it has been approved by the european commissionfor treatment of soft tissue sarcoma and ovarian

cancer, and it is pending fda approval forthe treatment of soft tissue sarcoma. another example is halaven, or eribulin, which isa fully synthetic analog of the marine sponge natural product halichondrin b. eribulin preventscell division, leading to cancer cell death, and it was approved by the fda in 2010 totreat patients with metastatic breast cancer that had already received two rounds of chemotherapybefore that. in the european commission, they actually approved it based on the resultsthat they had seen to treat patients that have received only one round of treatment,and they're actually looking into making it the first type of response that they willuse for breast cancer because they have seen a lot of very good results with this drug.i'm part of the marine biomedical and biotechnology

research here at harbor branch. our missionis to harness the power of marine biotechnology to improve the human condition through thediscovery of better drugs, alternative fuels, and industrial chemicals. we are composedof three groups. we have a natural products chemistry group that is led by dr. amy wright,and this group does the isolation and purification of secondary metabolites. they do the chemicalcharacterization of those compounds, so they figure out the structures, and they have worldwidecollaboration to test against dreaded diseases, from infectious disease to many differentkinds of cancer. we also have a marine microorganisms group that is led by dr. peter mccarthy. thisdoes the isolation and culture of marine microorganisms, and they also do biotechnology applications.they're trying to find enzymes to produce

alternative fuels, and things like that. mygroup is the cancer cell biology group, and so what we do is we screen these compoundsfor bioactivity, and, once we have an activity, we try to figure out how the compound doesits activity or the mechanism of action, so you will hear that phrase quite a bit. becauseat some point i'm going to toss these words at you, i wanted to introduce these conceptsso that you know what i'm talking about. the two key concepts in cell biology are apoptosisand signal transduction. apoptosis stands for a programmed cell death. it is a normalprocess in which a cell that has become infected or has finished its function, or is an oldercell, can kill itself in a very controlled way. this, as i said, is a very normal process,but, in cancer, this process is disrupted

and it allows cells that should be dying tosurvive. most cancer cells are resistant to apoptosis. one easy way to think of apoptosisis: we all have experienced infection, and, normally, when you have an infection, oneof the ways the doctors know about this is because your white blood cell counts are elevated,and that's because your white blood cells are responding to that infectious agent andthey're trying to destroy it. but, once they've done that, they kill themselves to eliminatethe excess of t-cells or white cells to just go back to normal because you don't need themanymore, so that's one easy way to think about apoptosis. now i'm going to move on to signaltransduction. in signal transduction, wikipedia says inbiology it's simply the process by which one

cell changes one kind of a stimulus into somethingelse, and cells kind of use signaling cascades. they activate something, that activates somethingelse, that activates something else, to do their function, so that's how they divide,that's how they produce factors that allow them to grow, to produce new blood vessels,that's how they undergo apoptosis, through these signaling cascades. amy came up withthe best way to think about signal transduction. if you call to order a pizza, the deliveryguy gets into his car and he follows all the signaling, and everything works out, you gotyour pizza. everybody happy. however, if he gets in his car and he tries to follow thesignals but he gets to a signal that is not working, what's going to happen is that hegets into an accident, no pizza, this is bad.

[laughter]if we put that in terms of a cell now, a normal cell, all the signaling pathways work, sowe get pizza, so only proliferates when it's needed, it can undergo programmed cell death,and it only grows in its tissue of origin. a cancer cell has defective signaling pathways,so those lights don't work. there's proliferation signals that are always on, the cells cannotundergo apoptosis, and this cell is able to grow in other tissues, and that's a characteristicof cancer ? a skin cell should never like to be growing in your lung, you know, so cancercells are very abnormal that way. i've been telling you about cancer, but ialways like to make this point: that there's not a signal disease that is cancer. canceris a collective name that we give to a group

of more than 100 diseases. what they havein common is that you have cells that are growing uncontrollably, and that these cellscontain damaged dna. the way they acquired that damaged dna could be through inheritance,it could be exposure to chemicals, it could be infections, it could be smoking, it couldbe excessive sun exposure, so there are many factors that could cause that. we believein the two-hit theory there's two events that normally lead to the cancer to start. my groupfocuses on pancreatic cancer, and the reason we focus on this disease is that it is thefourth leading cause of cancer death in the u.s. patients that are diagnosed, only sixpercent survive five years past that diagnosis, so the drugs that we're using currently arenot very effective. we have made progress

with many cancers in which we have reducedthe incidence and the lethality of many cancers, including breast and prostate cancer, butthe incidence and lethality rates for pancreatic cancer have been increasing in the last decade,so we really need new treatments to treat this disease.some characteristics of pancreatic cancer: we know that there are no early-detectionmethods currently exist. we don't have an easy way to diagnose it. the tumors are highlymetastatic. that means they spread to other organs. the tumors are resistant to apoptosis,and because most chemotherapies work by inducing apoptosis, the tumors are resistant to chemotherapy.we know that pancreatitis or inflammation of the pancreas seems to increase the riskof developing pancreatic cancer. we know that

pancreatic cancer cells are able to activatevery unusual pathways to provide nutrients to the growing tumor, and we know that pancreaticcancer uses the microenvironment surrounding it to grow. this leads us to put togethera model of pancreatic cancer progression. if you have a pancreatic cell and you exposethis cell to chronic inflammation, what's going to happen is you're going to activatesignaling pathways, such as the stat3 and nf kappa b that promotes survival and proliferation,and you're going to cause these cells to release soluble factors such as il-8 and ccl2. thesefactors attract cells of the immune system, and the cells of the immune system make itto the pancreas where they themselves release soluble factors. they release angiogenic factors,factors that promote the growth of new blood

vessels, and they release growth factors,creating a microenvironment that is very rich and that facilitates cells that are normalto grow. chronic inflammation also causes cell turnover, and that increases the chancesof a mutation occurring, so if you have a mutation occurring in these microenvironment,you're going to lead to a cancer cell. once you get one cancer cell, this microenvironmentis going to allow it to grow and form a tumor. now i'm going to put it in a scary form, soplease bear with me and i will explain this. we like to think disturbs. i told you inflammationcauses the release of soluble factors ? now i added a few more ? but what we know theydo is they activate signal transduction pathways, such as nf kappa b, stat3, ras and rage. thesepathways help that inflammation to persist,

and they also activate cellular events, suchas proliferation, resistance to apoptosis, activate unusual pathways to create nutrients,such as macropinocytosis, autophagy, and the growth of new blood vessels, such as angiogenesis.they also activate pancreatic stellate cells. these are normal cells that are around thetumor but they release soluble factors that create that microenvironment. they attractcells of the immune system that, again, help to perpetrate that inflammation, and theyhelp to create that microenvironment. when you have all these events occurring, it leadsto tumor initiation, and then to metastasis. this is scary but, for us, this provides targetsthat we can go and say, "can we act at these different levels and find inhibitors for thisdisease?"

we were very lucky that for many years wehad access to a ship and a sub that allowed us to collect marine invertebrates. from these,our chemists made extracts, so if you have coffee or tea, you're making an extract. ourchemists used something more harsh than water to make these extracts, but then they make- it's a similar process. they fractionate these extracts, and you can think of everysingle peak that you look at here as a compound, to create what we call the peak library, andthese are enriched fractions that contain a mixture of two or three compounds. theysend these to my lab, where we do screening, and, once we find the bioactivity, we sendit back to the chemists who isolate the pure compound based on the bioactivity, and thenthey send it back to us so we figure out how

it works or do mechanism of action studies.we do what is called phenotypic-based screening. we're testing the ability of compounds tochange a signaling pathway. if something is always turned on in cancer, we try to seeif we can turn it off, and vice versa: if it's not turning on, can we restore that.all our assays are cell-based, and we follow what we call a forward chemical genetics approach.we know we're changing a phenotype; we don't know where in the pathway we're causing thatchange, so we have to do mechanism of action studies to figure that out. this is a busyslide, but i just want to make a point that we have three main areas that we focus inmy lab. we look at molecules that regulate cell proliferation and survival, and theseare some of the molecules that we look at.

we have molecules that regulate inflammation,and we have molecules that affect mast cell degranulation and migration. i'm going totell you a little bit about the progress that we've made in these three areas.first, we're going to start with the marine natural compounds that target molecules thatregulate the inflammation. inflammation is a very normal response. it's how your bodytells your immune system that you have got an infection or that you have got an injury,so you want that to be there. it's only when it becomes what we call chronic or constantthat it becomes a problem, when it is not resolved, because when it becomes chronic,then you get tissue injury through the swelling, you get a microenvironment that facilitatestumor growth, and we have constant damage

and repair, which increase cellular turnover,which increases the probability of getting a malignancy. most adult cancers are frequentlypreceded by inflammation. long-term users of nsaids, such as aspirin, have a 40 to 50percent reduced risk of developing colon, lung, and stomach cancers, and we know thatpatients with pancreatitis have an increased risk of developing pancreatic cancer. thosethat have hereditary pancreatitis have a 53 times higher risk of developing pancreaticcancer than the average population, and those that have sporadic pancreatitis have about17 times higher risk than the normal population. i told you that inflammation causes the releaseof all of this: soluble factors, and activation of all these signaling pathways, but, forus, those represent targets that we can go

after. the first thing we want to see is canwe find inhibitors of il-8, and so the way we did this is we have pancreatic cancer cellsin the lab - the bxpc-3 - that produce high levels of il-8 without us doing anything tothem. i plated those cells and allowed them to adhere overnight. the next day, i replacedthe media with fresh media and the presence or absence of marine compounds. i incubatethese cells for about five hours to allow for new il-8 production, and then i take themedia where the il-8 has been released and i can measure that through an elisa, and thenanalyze the cells and mix it with ethidium bromide. that gives us an idea of the dnacontent in those cells, and we use that as a measurement of cell viability.an elisa is an enzyme-linked immunosorbent

assay, and the way it works is that we coata plate with a capture antibody. this traps the cytokine or soluble factor, and then weuse a different antibody, which is called a detection antibody, that also recognizesthe soluble factor, to kind of create a sandwich. once we have that, we can use horseradishperoxidase and a mixture of substrate that produces a color change, and we can followthat in a plate reader. that's a way to quantitate how much of that soluble factor or cytokinewas made. through that effort, we found theopederin k. this is the structure. it's quite complicated.it comes from the marine sponge discodermia. it's a known compound, so we knew that ithas had cytotoxic activity against tumor cells, but the inhibition of interleukin 8 is a newactivity for this compound. the ic50, or the

dose to see 50 percent inhibition for interleukin8, or for theopederin k is 370 nanomolar. the ic50 for il-8 inhibition for curcuminis 28 micromolar, so curcumin is a compound that is contained within the spice turmeric,and it is a known anti-inflammatory compound, but you can see how much more active thismarine compound is than curcumin, so we're pretty excited about that.the reason we're excited is because interleukin 8 promotes the growth of new blood vessels,and it also facilitates metastasis, so the inhibitors of il-8 may be anti-metastaticand anti-inflammatory. we also have an assay to screen for inhibitors of nf kappa b, whichis an important factor in regulation of inflammation and apoptosis. the way we do this is we havean engineered lung cancer cell line that contains

luciferase reporter gene for nf kappa b activity,so we give the tnf alpha to the cells, which is going to cause activation of the nf kappab pathway and nf kappa b subtranscription factor. that just means it makes it producegenes, and so it will produce all its normal genes, but it will also produce luciferin.when we combine that with luciferase, it produces light that we can follow through a plate reader.if we have an inhibitor there will be a decrease in increase that light, we can find it. throughthis assay, we found spongiatriol. spongiatriol has this structure. it comesfrom the spongia sponge. it has known anti-tumor and anti-viral activities, but inhibitionof nf kappa b is a novel activity for this. these here are cancer cells. this is flowcytometry results. but if you look at this

line, anything to the right of that line arecells that have active nf kappa b, and these are pancreatic cancer cells that have activenf kappa b. but if i treat them with two times the ic50 for spongiatriol, you can see thati have a lot less of the active nf kappa b. now, nf kappa b is important because it notonly regulates inflammation but, also, it blocks apoptosis from occurring. we knew thatpancreatic cancer cells have consistently levels of active nf kappa b in them, and soif we were blocking nf kappa b, we should be able to induce apoptosis in these highly-resistantcells. indeed, spongiatriol is able to induce apoptosis in two types of pancreatic cancercells, so that's very exciting results. through this type of assay, we also found microsclerodermina. microsclerodermin a has a much more complicated

structure, and it comes from a deep-watersponge of the genus microscleroderma. it has anti-tumor/anti-fungal activity but, again,inhibition of nf kappa b is a total novel activity for this compound. it has an ic50that is 1.2 micromolar, and it induces apoptosis in aspc-1, bxpc-3, and panc-1 cells, so thisone is also able to induce apoptosis in all our resistant cell lines.i keep on talking to you about apoptosis, and i think i should show you what it lookslike. we were very lucky that our management about six months ago got us a high-contentimager that allows us to take images very fast of our cells, and also quantitate differentcellular events. through generous donations, i had the means to buy fluorescent reagentsthat allows us to capture these events. these

are pancreatic cancer cells. they have beenlabeled, their nuclei, in blue. if they are undergoing apoptosis, they are going to cleavea molecule called caspase-3, and it's going to look green. if they die, their membranewill lose its integrity and they're going to be able to absorb a red dye, so apoptoticcells are going to look blue, then green, then red. we took pictures every 20 minutesfor 16 hours, and you can see that there's a few cells undergoing apoptosis. this isour methanol vehicle control. now i'm going to show you what microsclerodermin a doesin these cells. again, remember apoptotic cells, blue, then green, then red. we're veryexcited about that. you can see how much active our compound is, and i wanted to use thatas my christmas card, but -

[laughter]- people thought it was too geeky. for inhibitors of inflammation, we have so far, for nf kappab, we have found 9 pure compounds that inhibit these pathways, and 70 fractions correspondingto 23 different organisms that require further purification. for stat-3, we have found 20fractions corresponding to 15 different organisms that require further purification. for interleukin8, we have found 10 pure compounds that are able to inhibit il-8. we're currently screeningfor rage, which is a receptor that's involved in regulation of inflammation. the screeningis ongoing but, so far, manzamine a is kind of showing activity, and i'm going to tellyou a lot about manzamine a, and, hopefully, you will be as excited about this compoundas i am.

our conclusions for inhibitors of inflammationin pancreatic cancer: inflammation plays an important role in both the initiation andprogression of pancreatic cancer. we have found marine natural compounds that are ableto inhibit important inflammatory signal transduction pathways. these compounds have the potentialto not only treat pancreatic cancer, but also to prevent it from ever forming if it's givento patients with pancreatitis. so we're very excited about these compounds, and we're hopingto submit a larger grant to screen for more active compounds, and also to further ourunderstanding of the compounds that we already have found. now i'm going to tell you aboutmarine natural compounds that target mast cell degranulation and migration.mast cells are very important in pancreatic

cancer. when you have a pancreatic tumor,it's normally surrounded by what we call the stroma, which is made out of connective tissueand healthy neighboring cells, but they contribute to that microenvironment that kind of facilitatesthe tumor growth. we know that the tumor cells release ccl2, and that attracts mast cellsto migrate to the tumor stroma. once the mast cells get there, they degranulate and theyrelease molecules that cause blood vessel formation and that favor proliferation, andthis, in turn, help the tumor to grow and metastasize. again, when i present these pathways,we see opportunities to target things, and so we thought about finding compounds thatblock the release of ccl2 to prevent the mast cells from ever migrating to the pancreas,or finding compounds that block degranulation

to prevent the release of those factors. thosecompounds will also have utilities for other diseases because mast cells, for those ofyou that suffer allergies, they are the ones responsible. they're the ones that releasethe histamine that gives you your runny nose and your watery eyes.the first thing that we did was create a ccl2 elisa to try to find inhibitors of this. weused a different kind of pancreatic cancer cell line because this one produces largelevels of ccl2 just by themselves. we don't have to do anything to them. we plated thecells and we allowed them to adhere overnight. the next day, we replaced the media with freshmedia in the presence or absence of marine compounds. we incubate for about five hoursto allow for new ccl2 production, and then

we take that media and measure how much ccl2was produced through an elisa assay, and again, we measured viability by measuring the dnain the cells. that has led us to find the compound discodermide. discodermide is a compoundfrom the sponge discodermia dissoluta. it has known cytotoxic and anti-fungal activities,but inhibition of ccl2 secretion by pancreatic cancer cells is a total novel activity forthis compound. the ic50 or the dose required to see 50 percent inhibition for ccl2 of discodermideis 10 micromolar. the ic50 for ccl2 inhibition of curcumin is 45 micromolar. again, the marinecompound is far more active than the compound that is known to have anti-inflammatory activities.inhibitors of ccl2 may prevent the mast cells from migrating to the site where the pancreatictumor is growing.

we also have an assay for finding inhibitorsof degranulation. i told you that the mast cells release many soluble factors in additionto the growth factors and angiogenic factors. they release histamine, and they release thisenzyme called beta- hexosaminidase. we can induce degranulation by adding the chemicalcalcimycin, and we hope that in the presence of harbor branch compounds, we will see alot less degranulation. we can take the beta-hexosaminidase that was produced, and we mix that with adifferent compound and that produced a light change that we can follow through our platereader. that allowed us to find a new activity for the compound topsentin. topsentin is acompound that we've been very excited about. it has a cytotoxic activity against tumorcells and it has anti-viral activities. we

knew that it was a potent anti-inflammatorycompound, both in b1 and b2, but we didn't know that it acted upon mast cells, and thatmakes it very exciting because mast cell influx is not only important for pancreatic cancer,but it has been seen with many other kinds of cancer, so this has potential to be veryeffective against many different cancers. the ic50 for inhibition of degranulation fortopsentin is 11 micromolar. so far, we have found two pure compounds thatinhibit ccl2 secretion by pancreatic cancer cells, we have found 5 pure compounds thatinhibit mast cell degranulation, and 27 fractions corresponding to 12 different organisms thatrequire further purification. we know that mast cells play an important role in pancreaticcancer initiation and progression. we had

a small nih grant that allowed us to do thiswork, so we only did a discreet screening of about 500 compounds. our library containsabout 5,000, so we want to do more screening because there might be compounds that areeven more active than the ones that we have found so far, but the good news is that wehave found compounds that inhibit ccl2 secretion by pancreatic cancer cells. we also have foundcompounds that inhibit mast cell degranulation, and these compounds have the potential toprevent pancreatic cancer from forming, and they also have the potential to treat pancreaticcancer that is already there. we're hoping to submit a larger grant to screen the compoundsthat we've already found and to find new ones sometime this year. now i'm going to tellyou about marine natural compounds that affect

cell proliferation and survival.the first compound that i'm going to tell you about is leiodermatolide, and some ofyou might have already heard about this compound because we're very excited about this compound.we try to keep you abreast of the research that we do with it. but this is a potent antimitoticagent that was isolated from the leiodermatium sponge. it's about tenfold more active thantaxol, and i told you taxol is the drug that gets used the most against cancer. i alsochose activity not only against pancreatic cancer cells but against triple-negative breastcancer and against some colon cancers. one of the ways that we figure out that it isantimitotic is by doing imaging, so these pancreatic cancer cells were labeled withan antibody for tubulin, which is the red,

and tubulin is very important. it gives thecell its shape, and it also is very important for cell division. you need functional tubulinfor that to happen. these cells were also labeled with an antibody for phospho-histoneh3, which can only be seen in cells when they're doing cell division, so this is our control.you can see that there's a few cells that are undergoing cell division but, when youtreat with leiodermatolide, you cause arrest in cell division and so you get to see manymore cells that are undergoing cell division because you basically stop that cell divisionfrom occurring, so we know leiodermatolide causes mitotic arrest.this the boring way which we used to look at apoptosis before we had our imager, butwe're looking at the same thing. cleavage

of molecules called caspases is a hallmarkfor apoptosis, and you can see that leiodermatolide cleaves a lot of the caspase-3 and 7 after24 hours of treatment. similarly, we look at dna fragmentation as a hallmark of apoptosis,and you can see that leiodermatolide indeed induces this dna fragmentation in pancreaticcancer cells. so we know that leiodermatolide induces apoptosis in pancreatic cancer cells.most compounds that cause cell cycle arrest do so by acting upon tubulin, and tubulinis constantly coming together and pulling apart. when it comes together, we call thattubulin polymerization; when it pulls apart, we call that depolymerization. most compoundsthat are antimitotic affect tubulin in one way, so here you have taxol, and taxol causestubulin polymerization, so you can see how

much higher that is. we have vincristine andnocodazole, which cause depolymerization, and you can see how they are down in the graphbecause they are depolymerized. but, if you have tubulin alone, if you have tubulin treatedwith a vehicle control, or tubulin treated with leiodermatolide going from 1 micromolarto 100 micromolar, we had no effects, so we know that leiodermatolide does not cause tubulinpolymerization or depolymerization. these are our mechanism of action studies.we know that leiodermatolide is a potent antimitotic compound. it has no effect on polymerizationor depolymerization of tubulin. we know it affects tubulin microtubule dynamics, butwe don't know exactly where. we haven't been successful at determining exact moleculartarget, if it's binding something, but everything

hints at a possible novel mechanism of action.so, the bad news is we still don't completely understand its mechanism of action, even thoughwe have tried a lot of things. the good news is that everything that we have tried tellsus that this compound is unlike any other compound that causes cell cycle arrest, sothat makes it quite unique, and so that is very exciting because it might have the potentialto work on those cancers where taxol doesn't work.the other good news is that we have a colleague at ucf that has a mouse model of pancreaticcancer, and they put leiodermatolide through this mouse model and, at the end of the day,at the end of their study, they measured tumor weight. you can see that leiodermatolide significantlydecreased tumor weight when you compare it

to control and when you compare it to animalsthat were treated with gemcitabine, and gemcitabine is the drug that is used to treat pancreaticcancer right now in the clinic, so these are good news. this tells us that leiodermatolidenot only works on cells, but it works in vivo, so this puts us just one step closer to aclinic, so we're very excited about these results. leiodermatolide so far, we know thatit is a potent antimitotic agent that was isolated from the leiodermatium sponge. it'sabout tenfold more active than taxol. it has potent cytotoxic activity against pancreaticcancer cells, against triple-negative breast cancer cells, and now against colon cancercells. because of cell cycle arrest by a very unique mode of action, it affects tubulindynamics and it has in vivi and in vitro anti-tumor

effects. now i'm going to talk to you aboutmy baby. i have ugly babies. this is the molecule, manzamine a, that i'vebeen working with since i came to harbor branch, and i am super excited about what this compoundcan do, so i hope that you will be, too, after i show you. manzamine a has been isolatedfrom xestospongia sponge and haliclona sponge, and we actually believe that it's a microbeinside those sponges that makes that compound. some of the reasons we got excited about thiscompound is that manzamine a abrogates cell dissociation.and you can see here cancer cells, and they're not touching each other. they like to liveon their own, and this is very abnormal. cells like to touch each other and get their signalsout of each other. look at your skin, and

look at how close they are. you can see thatthe cells alone, they are not touching much, they are not spread out, they kind of lookready to migrate, which is a bad sign. it's a characteristic of a metastatic cell. ifyou treat these cells with uo126, which is an inhibitor of p-mek, which is part of theras pathway that i showed you earlier, you can restore the association of the cells,you can see that now they're spread out, they're touching each other, but you get similar effectsif you treat with 2.5 micromolar of manzamine a, and it's much more apparent with 5 micromolarand 10 micromolar of manzamine a, which is about one-fifth of the dose of the other inhibitor.i keep on telling you about apoptosis and our cells being resistant to it, so when youhave a normal cell, they have what we call

death receptors. when you bind that receptorwith its ligand, in this case a tnf receptor ? apoptosis-inducing ligand, or trail ? youwill induce apoptosis. if you have a cancer cell, though, you can bind the death receptorwith trail, and you still get survival. what we were hoping is can we put or pre-treatwith our harbor branch compound and then bind with trail, and see if we can restore theability to undergo apoptosis. this is a very busy slide, but what i want you to focus ison this side where the apoptosis is occurring, so in this quadrant you want to look at theright, the top has the late apoptotic cells, and the bottom has the early apoptotic cells.you can see that when i treat with manzamine a, i get about 35 percent apoptosis for thesepancreatic cancer cells. when i treat with

trail and methanol, again, i get about 35percent apoptosis, but, if i treat with a combination of 10 micromolar of manzaminea and trail, i get about 85 percent apoptosis, so manzamine a is not killing the cells itselfbut it restoring the ability of the cells to undergo apoptosis. this is very exciting.next, because we had seen the abrogation of cell dissociation, we wanted to see if ithad other anti-metastatic effects. we know that cells that are metastatic are able tomigrate to other organs, and so we can mimic that in the lab by creating this transwellexperiment where we put an insert in our subculture plate. we can coat that insert with a collagenmatrix, and we create what we call a chemotactic gradient by just putting more fbs. we plateour cells on the top, and then we watch them

if they make it to the bottom of the plate,so we kind of follow the cells when they're here. you can see that the cells that areuntreated or just receiving methanol are very able to migrate to a membrane alone. theyhave a little bit more difficulty when we have collagen in there, but they still madeit. if we treated with uo126 or with manzamine a, we took away the ability to migrate. thattranslates to anti-metastatic properties, so that made us very, very excited.we knew manzamine a had all these great abilities, but we didn't know how it worked, so we hadto start doing mechanism of action studies. dr. amy wright at that point had a grant whereshe was collaborating with novartis, and they put manzamine a through the hip-hop assay.hip stands for haploinsufficiency profiling.

you have yeast strains, which have a singlecopy of a gene knocked out. every gene has two copies, so we just took down one, andthe strains that are most sensitive to the compound that they're being exposed will becomedepleted over time, and it's illustrated over here. you can see the greens have gone away.through this assay, you can identify the compound started, so if the compound binds something,we will identify it through the hip. we can also do homozygous profiling, where now weknock both copies of genes that are not essential, and the strains more sensitive to a drug,again, will become depleted, just like this. this will tell us which genes are requiredfor growth in the presence of the compound, and this is very informative for those compoundsthat lack a direct binding target.

this is complicated but i will try to simplifyit a little bit. this is manzamine a, and this is the hip profiling and this is thehop profiling. under here, we have prodigiosin prodigiosin is a compound that is very wellstudied, and we know it's known v-atpase inhibitor. you can see that the profiles almost match.we have no hits on the hip profiling, and we knew that manzamine a had no direct bindingtarget, but the hop cluster with known uncouplers of vacuolar atpases, and that gave us a hintthat that was manzamine a's mechanism of action. so, what does that mean? well, if a cell hasuncouplers of vacuolar atpases, one of the possible results would be that you will accumulateacidic autolysosomes. and so my post-doc followed this, and he did observe that you have that.so here's our vehicle control, and the more

green they are the more brilliant, the moreacidic they are. here, you have cells treated with manzamine a, this is our close-up at40x, and you can see that there's some accumulation of acidic autolysosomes after treatment withmanzamine a, and this is very similar to bafilomycin a1 treated cells, that this is a known uncouplerof vacuolar atpases. surprisingly, the combination of both didn'tcause even further fluorescence, so we don't know exactly what is going on there. it willrequire a little bit more studies. but, this confirmed that, indeed, manzamine a uncoupledvacuolar atpases, and this led george to think, "okay, does that have an effect on autophagy?"an autophagy is a pathway that only gets activated under stress, specifically if you're understarvation. if a cell is undergoing starvation,

what it does is it pulls out any organellethat it's not using and puts it in a vesicle, then that gets broken down with an autolysosometo produce amino acids and fatty acids, and that allows for new protein synthesis andenergy production, and the cell is able to survive. think if you were trapped in a cabinduring a blizzard, you will burn anything you need to keep warm, so this is what thecell does. but what cancer does ? very smart ? it grabs the neighbor healthy cell and breaksit down through this process to obtain nutrients. george wanted to find out what happens whenwe treat cells to autophagy in this pathway, and so we were able to follow lc 3-ii, whichis a marker of autophagy, that basically the levels increase as autophagy is occurring,and they resolve as autophagy resolves. you

can see this western blot for lc 3-ii, andthis lc 3-ii at 24 hours. we got very high levels of it. by 48 hours, they still keepon increasing. and you see under starvation, we induced autophagy to occur, and we sawthe increase in lc 3-ii, but, by 48 hours, it had already resolved. so these led us toconclude that manzamine a was inhibiting autophagy from occurring. we know that manzamine a isnot very cytotoxic on its own but it has potential anti-metastatic activities because it preventsmigration through a collagen matrix. it also reduces cell dissociation. it restores theability of pancreatic cancer cells to undergo apoptosis. it uncouples vacuolar atpases,increasing lysosomal acidity, and inhibits autophagy.if you recall that pathway that i showed you,

that scary pathway at the beginning. so weknow inflammation can activate all the signaling pathways, which do all these activities, leadtumor initiation and metastases. i put manzamine a here in gold because we know that manzaminea inhibits nf kappa b, it appears to inhibit rage. we know it abrogates apoptosis resistance,we know it inhibits autophagy, and it seems to have anti-metastatic properties, so thiscompound is attacking pancreatic cancer through five different ways, so we are super excitedabout this compound. we hope that this is a compound that makes it to clinic, and that'swhy i call it my baby because i'm super excited about the abilities of this compound.i'm going to tell you a little bit of future directions that we're planning to go. whenwe have a cancer cell, how does it go into

a full-grown tumor? there's a lot of eventsthat have to happen, but with pancreatic cancer, we knew we could block angiogenesis and thetumor growth would still occur, we could cut certain things and it still managed to grow,and that led us to understand that pancreatic cancer is able activate very unusual pathways.one of the pathways that it activates is macropinocytosis. we like our big names. what happens here isthat the cancer cell uses its membrane to trap proteins to create this cup, to trapproteins that are outside of the cell, bring it inside the cell, then it fuses the vesiclewith the lysosome, where it gets broken down into amino acids, and that promotes tumorgrowth. we also know that pancreatic cancer likes to activate autophagy, where it canisolate organelles into an autophagosome,

and then this, again, fuses with a lysosome,they get broken down, they produce amino acids, and produce this tumor growth. as i said,these organelles are likely to be out of the neighboring healthy cell.again, we see targets, so we are trying to find compounds that block macropinocytosis,and we're trying to find other compounds in addition to manzamine a that might block autophagy.autophagy is going to be important not just for what i told you, but this was a big headlineon the scientist, which is a publication that we get, that came out after the last meetingof the american association for cancer research. what they had found is that in those cellsthat responded to chemotherapy and they were starting to undergo apoptosis, if those cellsactivated autophagy, they were able to thwart

the chemotherapy and survive by activatingthis pathway, so inhibitors of autophagy are going to be very important in fighting cancer.for macropinocytosis, we have pancreatic cancer cells that we know can activate this pathway,so we're basically just letting it do it, but we're putting a fluorescent tag for itto ingest. once in there, we are going to follow it using our high-content imager thatcan image and quantitate the cells, and this is what that looks like.these are pancreatic cancer cells that were labeled with blue for the nuclei, with greenfor their body, and those that have internalized the fluorescent tag look kind of pinkish,so you can see all those that have arrows have internalized the fluorescent tag thatwe put. the beauty of a high-content imager

is that it quantified this for me, and sowe get something that looks like this. we know that our pancreatic cancer cells by themselveshave some basal levels of macropinocytosis, but if i put them under conditions of serumstarvation, i can really induce a lot of it. if i treat them with manzamine a, it bringsthat down, so this is a novel activity for manzamine a. i need to confirm it, but it'svery exciting. eipa is a known inhibitor of macropinocytosis. it didn't work that greaton this assay. i'm still trying to optimize this assay, but we're very close to havingan assay that we can screen for inhibitors of macropinocytosis.for autophagy, we're following a very similar approach to what george did. we are lookingat lc3ii, and so this is how our staining

looks. again, cells are labeled in blue withtheir nuclei, green for their body, and you can see the red lc32 staining on their membranes.this is a non-specific antibody, and i don't like this differential, so i'm still tryingto optimize this staining, which is hopefully going to happen sometime soon. our conclusionis that we are setting up assays to identify inhibitors of autophagy and macropinocytosis.a discreet screening of about 500 fractions will occur this year, and we hope that compoundsthat inhibit these pathways will cut the fuel lines of pancreatic cancer so they will preventthe tumors from growing. and we hope to acquire sufficient data to submit an nih grant.to recap, i know i've given you a lot of information, but if you remember these last two slides,you will be okay. i gave you the scary pathway

of progression of pancreatic cancer, so thisis inflammation. we know it releases a lot of factors, it activates transcription factors,it activates pancreatic stellate cells, and it attracts cells of the immune system. itactivates many cellular events that lead to tumor initiation and metastasis. hopefully,i have convinced you that we have many different marine natural compounds that are able toinhibit these different events earlier on, and what we hope that will happen is thatthese compounds can then inhibit all these cellular events that lead to tumor initiationand metastasis. i just want to acknowledge everybody thatworked to get this. george was my former post-doc. he did the work for the mechanism of actionof manzamine a, and he also helped set up

the degranulation assay. tara, dee, and patare staff scientists mbbr. they did the bulk of the screening for all the assays that ishowed you, and also assist me in the mechanism of action studies. mike, gervaise, lexi, andkelly are former summer interns whose work has contributed to these results. and we haveour collaborators at novartis ? dominic hoepfner and tiphaine jaeg. they ran the hip-hop assayfor manzamine a. dr. cheryl baker at ucf, ran our animal studies for leiodermatolide.i also want to acknowledge all our funding sources. we are self-funded, so we have nihgrants that support our work. i also got a florida department of health grant that allowedus to do a lot of the inflammation work. we are part of the cooperative institute thatruns from noaa that shirley pomponi runs here

at harbor branch, and that has allowed usto follow some of our mechanism of action studies, and we hope that it's going to helpus collect new samples. the baernard a. egan foundation, the roseann gregory cancer foundation,and the lloyd biddle popcorn club have made generous donations to my research.it's thanks to these donations that i'm able to do those experiments that just take myresearch one step above. the images that i showed you about apoptosis were submittedas part of my publication to support the data that i had, but they also allow us to follownew directions, so any time that we find something new about pancreatic cancer, it's thanks tothose donations that i'm able to set up assays and try to find those inhibitors. i also wantto thank everybody that is part of the mbbr.

we're truly a multidisciplinary and collaborativegroup, so everybody is involved in the effort and the research that i showed you. with that,i thank you and i'll take any questions.