| Ovarian cancer is the fifth lethal cancer of female reproduction systerm malignancies in America, the high mortility dues to the late stage diagnosis when the cancer cells spread outside of the ovaries. Although good prognosis of the surgery following by chemotherapy could be achieved, but just 30% of the patients who could survive 5 years after the diagnosis, the challenage of the ovarian cancer is the lack of early detection methods and the metastasis and chemoresistance of late stage ovarian cancer cells, Detection in early stage and comphrehensive, safety and effective treatment strategies of ovarian cancer are two of the most important issues should be investigated in future.Genetics and epigenetics work together to determine the fate and progression of human cancer, the epigenetics is different from genetics, it is heritable and reverseble and could change the statue of the cell from malignancy to normal and vice versa. The disturbance and ablation of the epigenetic regulation involve in the mechanism of most human disease, With the development of the research of epigenetics of human cancer, especially the DNA methylation, histone methylation and histone acetylation and other modifications, the epigenetic realated enzymes and proteins would become the target of the cancer treatment.The disorder of the epigenetic statue could increase the instability of the genome and eventually cause cancer, So if the combination of the epigenetic modifiers and conventional chemotherapeutic reagents could make the cancer cell resensitizing to conventional chemotherapeutic reagents in ovarian cancer is unknown, but the application of the inhibitors of DNA methyltransferase, histone methyltransferase and histone deacetylase are becoming more and more in preclinical and clinical trials.We assume that the combination and sequential treatment strategies could affect human ovarian cancer in aspect of molecular biology, cellular biology, morphology and tumorgenicity and would provide us a promising treatment strategy for ovarian cancer patients.Chapter 1 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell lineOBJECTIVE:To investigate the effect of the single or combination of epigenetic modifiers and conventional chemotherapeutic reagents on the tumor biology, epigenetic regulation, spheroid formation and growth of Hey cells, and to find the detailed mechanism of the response of Hey to epigenetic modification and try to figure out and improve the the management of metastasis and chemoresistance of the ovarain cancer.METHODS:1 Cell preparation and Isolation of protein1.1 Cell preparation:Cells (treated or untreated) are growing to 80-90% confluence and trypsinized after removing growth medium. Cells are washed once with PBS and pelleted by centrifugation for protein isolation.Total isolation of protein:Prepare 1X extraction buffer by adding 1 ml of 5X extraction buffer to 4 ml of distilled water. Add DTT solution and PIC to 1X extraction buffer at the 1:1000 ratios. Re-suspend cell pellet in 100μl of ice cold 1X extraction buffer per 106 adherent cells or 2 x 106 non-adherent cells. Transfer the cell solution to a microcentrifuge vial. Incubate on ice for 15 min with vigorous vortex 5 sec/5 min. Centrifuge the cell solution for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of cell extract. Use immediately or aliquot and freeze supernatant at -80℃until further use. Avoid repeated freezing/thawing.1.2 Isolation of histone:Harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 min at 4℃. Dilute 10X Pre-Lysis Buffer into 1X Pre-Lysis Buffer with distilled water at a 1:10 ratio. Re-suspend cells in the Diluted 1X Pre-Lysis Buffer at 106cells/ml and lyse cells on ice for 10min with gentle stirring. Centrifuge at 3000 rpm for 5 min at 4℃. If cell lysates are prepared in a 1.5ml to 2ml size vial, centrifuge at 10,000 rpm for lmin at 4℃, Remove supernatant. Re-suspend cell/tissue pellet in 3 volumes of Lysis Buffer and incubate on ice for 30 min. Centrifuge at 12,000 rpm for 5 min at 4℃and transfer the supernatant fraction (containing acid-soluble proteins) into a new vial. Prepare Balance-DTT Buffer by adding DTT Solution to Balance Buffer at a 1:500 ratio. Add 0.3 volumes of the Balance-DTT Buffer to the supernatant immediately. Quantify the protein concentration with an OD reader. BSA can be used as a standard. Aliquot and store the extract at -20℃for several days, or -80℃for long-term storage. Avoid repeated freezing/thawing.1.3 Nuclear extraction:Growing cells to 70-80% confluence on a culture plate or flask. Remove the growth medium and wash cells with PBS twice and then remove PBS. Add lml of fresh PBS per 20 cm2 area, and scrape cells into a 15ml conical tube. (Alternative option:detach cells with trypsin-EDTA and collect cells into a 15ml conical tube. Count cells in a hemacytometer). Centrifuge the cells for 5min at 1000 rpm and discard the supernatant. Dilute NE1 with distilled water at a 1:10 ratio. Add DTT solution and PIC to ice cold diluted NE1 (IX) at a 1:1000 ratio. Re-suspend cell pellet in 100μl of diluted NE1 per 106 cells and transfer to a micro centrifuge vial. Incubate on ice for 10min. Vortex vigorously for 10 sec and centrifuge the preparation for 1 min at 12,000 rpm. Carefully remove the cytoplasmic extract from the nuclear pellet. Add DTT solution and PIC to NE2 at a 1:1000 ratio. Add 2 volumes of NE2 containing DTT and PIC to nuclear pellet (about 10μl per 106 cells or per 2 mg tissues). Incubate the extract on ice for 15 min with vortex 5sec/ 3min. The extract (especially tissue extract) can be further sonicated for 3 X 10 sec to increase nuclear protein extraction. Centrifuge the suspension for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of the nuclear extract. Use immediately or aliquot and freeze the supernatant at -80℃until further use. Avoid repeated freezing/thawing.2 Western Blotting Protocol2.1 Making gel:Place a short plate on top of the spacer plate, Slide the two plates into the casting frame, keeping the short plate facing front. Insure both plates are flush at the bottom on a level surface. Lock the pressure cams to secure the glass plates. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand gasket. Insure the horizontal ribs on the back of the casting frame are flush against the face of the casting stand and the glass plates are perpendicular to the level surface. The lever pushes the Spacer Plate down against the gray rubber gaskets. Place comb in between glass plates. Only put teeth in between glass. Mark 1 cm below where teeth are on outside of glass plate. Pour separating gel into 50 ml beaker:see recipes below, Take up 7 ml of gel using 5 ml pipet. Put pipet on edge of glass and hold at a 90 degree angle and begin filling to mark made on glass plate. Add 500ul of H2O. Let sit for 45 minutes. When gel is polymerized, invert apparatus and empty H2O. Pour in stacking gel. Place comb into gel. Let sit for 45 minutes. Remove the gel cassette sandwich from the casting frame and place it into the electrode assembly with the short plate facing inward. Slide the gel cassette sandwich and electrode assembly into the clamping frame. Press down the electrode assembly while closing the two cam levers of the clamping frame. Lower into the mini tank and add 400mls of lx Running Buffer.2.2 Sample preparation:Dilute sample if necessary in dilution buffer below (or RIP A),For whole cell preps, typically use 50-100ug of protein per lane (20-50ug for nuclear,50-100 for histone), For each sample, add desired amount of protein to a microcentrifuge tube followed by 6x SDS Sample buffer or 2x Laemmli. Note:BME should be added fresh to the loading buffer, For the protein markers, add 5ul of markers to tube and lul of 6x sample buffer or 5ul of 2x Laemmli buffer. (Note:if you want to be able to see the marker while the gel is running, load 7-8ul instead of 5ul. Incubate at 95-100℃for 3 minutes. Quick spin the sample and load on gel.2.3 Running gel:Once gel is polymerized, remove gel sandwich from casting stand and insert into inner cooling core of running rig. Fill inner core with 1X running buffer. Fill outer chamber with 1X running buffer until half of the screws are covered. (make 400ml for mini rig,500ml/gel for Criterion), Blow out wells. Load samples. Run at 75 volts for 1-2 hours or longer depending on the amount of separation. Before gel is done running, get out 2-3 pyrex dishes and fill with transfer buffer. Soak 2 filter papers and 2 fiber pads per gel for at least 5 minutes. Soak membrane in transfer buffer for at least 5 minutes. Stop electrophoresis.2.4 Membrane transfer:Remove gel. Separate plates. Use spacers to separate. For Criterion rig, use lid to help pop open the cassette. Cut off stacking gel/wells with razor or spacers. Put plates into transfer buffer and nudge gel off plate with spatula into the transfer buffer, Soak gel for 10 minutes. Open sandwich with black side down and stack in this order:fiber pad, filter paper, gel, membrane, filter paper, fiber pad. Make sure to roll out bubbles after gel, membrane and second fiter paper. Close and put into transfer chamber. Black to back. Put in frozen block. Fill transfer chamber with transfer buffer. Transfer at 30V overnight in the cold room or at 100V for 1-2 hours on bench, changing ice pack half way through if needed.2.5 Washes and detection:Open sandwich. Put membrane in clean container. Rinse membrane with dH2O 2-4x. Block with 50mls 1 hr RT (Blocking buffer:5% non-fat dry milk in TBS-T), Wash with TBS-T 3×10min at RT, Add primary antibody and incubate for 1 hour at RT or overnight at 4C. Wash in TBS-T 3×10 minutes at RT. Add secondary antibody at 1:1000 in TBST with 5% milk and incubate for 1 hour at RT. Wash in TBS-T 3×10min at RT. Drip dry the membrane onto a paper towel and lie flat on a piece of Saran Wrap with the protein side facing up. Mix 3 mls of chemiluminescent substrate Reagent 1:1 and pour onto the membrane. Let incubate for 1 minute. Dump solution off of membrane and drip dry on paper towel. Cover membrane with saran wrap. Expose on G:box for 1 minute to 5 minute depending on the strength of signal.3 Spheroid formation assayThe cells are cultured in 6/12/24/96 wells ultra-low attachment plates at a concentration of 500 cells/ml in 2 ml/well Complete MammoCultTM Medium in a humidified incubator. A minority population of cells will began to grow as multi-cellular spheres, as opposed to the majority of cells which will not survive. Cells are examined microscopically on a daily basis and growth rates recorded. All the treatment proceure are accroding to the experiment schematic representation and all the spheroid would be taken pictures and assayed in aspect of size, cell number, spheroid number and cell viability. RESULTS:1 The effect of the treatment on the tumor biology of Hey cellsOur data indicated that no E-cadherin has been detected in Hey cells, but strongly expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could slightly increased the expression of E-cadherin, but not E-cadherin. The combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could not change the E-cadherin expression. There is no significanlty change of Pan-AKT and AKT-1 in presence of drugs, The combination of Decitabine/Cisplatin could significantly suppress the ABCG2, but not found in other treatment group. The pluripotency markers OCT4 strongly expression in Hey cells, but moderate expression of NANOG and SOX2, No effect of treatment on the expression of these pluripotency markers. Mitochondrial apoptosis pathway protein Bax has been increased after the single drug treatment. Our data indicated that strongly E-cadherin has been detected in Hey cells, but weak expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could not increase the expression of E-cadherin, but the combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly change the E-cadherin and N-cadherin expression, Trichostatin A and decitabine could increase AKT1, but the combination of decitabine/Cisplatin could suppress the AKT1,All the treatment significantly suppress the ABCG2, Decitabine/Cisplatin suppress the expression of NANOG, but SOX2 and OCT4could be affected by both combiantion, Bax could be suppressed by Trichostatin A/Cisplatin and Decitabine/Cisplatin.2 Effect of the treatment on epigenetic regulation of Hey cell lineDNMT3a and DNMT3b could be detected in Hey cells and Cisplatin could slightly increase the expression of DNMT3a, but not of DNMT3b, No effect on LSD1 and histone methylation markers. DNMT3a is strongly expression in SKOV3 cells but slightly expression of DNMT3b, Trichostatin A, Decitabine and Cisplatin could decrease the expression of DNMT3a, but Decitabine/Cisplatin has more suppress effect on DNMT3a, Interestingly, Trichostatin A/Cisplatin and Decitabine/Cisplatin could totally suppress LSD1. The effect of drugs on the histone methylation is diversity and complexity.3 Effect of the treatment on spheroid formationThe Hey spheroid is big and compact in compare with the IOSE, SKOV3 and A2780. Interestingly, the A2780 spheroid is loose and out of shape, the formation of the spheroid has the features of time and dose response in presence of the drugs, and the combination of Trichostatin/Cisplatin and Decitabine/Cisplatin could totally suppress the shperoid formation. The combination strategies also could suppress the spheroid growth and the number of spheroid formation. The Spheroid formation of SKOV3 in presence of different treatment strategies has time and dose dependent effect and all the strategies significantly suppress the formation of Spheroid, in the spheroid growth assay. Trichostatin A, Cisplatin 5um and Trichostatin A/Cisplatin totally suppressed the Spheroid growth and Spheroid cell viability, but Decitabine,Cisplatin lum and Decitabine/Cisplatin has no significant effect, The combination of Trichostatin A/Cisplatin could suppress the Spheroid number in 8-fold in compare with control group. So the combination could achieve great Spheroid inhibition effect.CONCLUSION:High expression of N-cadherin could be detected in Hey cell line and the it could be changed in presence of drugs alone or combination, the ABCG2 has been significantly suppressed by the decitabine/CSP combination and pluripotent markers OCT4/NANOG/SOX2 highly express. No significantly inhibition of DNMT and LSD1 and even the histone methylation marks, TSA/CSP blcoked the formation and growth of spheroid, and the optimal strategy could not only kill the cancer cells but also have the protection effect of normal epithelial surficial ovarain cells. The combination of Trichostatin, Decitabine and Cisplatin could significantly supppress the expression of E-cadherin and N-cadherin in SKOV3 cells, The ABCG2 could be totally suppressed by all treatment. Strongly expression of pluripotency markers OCT/NANOG/SOX2 could be found, No effect has been detected on the mitochondrial apoptosis pathway, DNMT 3a was highly expressed but not of DNMT3a, Interestingly, The Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly suppress the LSD1. But the expression of histone methylation markers showed more diversity and complexity, Methylation of H3k4 and H3K9 reduced in presence of Trichostatin A/Cisplatin. Moreover, Drug alone or combination could totally suppress the formation and growth of spheroid of SKOV3.Chapter 2 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell line Hey tumorigenicity in mouse modelOBJECTIVE:To establish the animal model of ovarian tumor formation, growth, abdominal metastasis and the effect of treatment of combination and sequential on tummorgenecity and behavior of ovarain cancer, try to find a promising strategy to cure human ovarain cancer.METHODS1 Preparation of tumor cells Growing cells in complete medium and when cells reach 70-80% confluent, replace medium with fresh medium to remove dead and detached cells when 3-4 hrs before harvesting. Remove medium and wash cells with could PBS. Add a minimum amount of trypsin-EDTA. Disperse cells and add complete medium (10:1 to 5:1). Centrifuge immediately at 1500 rpm for 2-5 min and wash twice with PBS and store cells. Count cells using a hemocytometer. Using typan blue staining to exclude dead cells. Mix cells 1:1 with typan blue solution. Cells should be suspended in a volume of 300μl contains required number of cells per injection.10×106 cells are needed per injection per mouse.2 Preparation of mice. Mice should be 4-6 weeks old. Allow 3-5days acclimatization period after mice have arrived.3 Preparation of injection Clean and sterilize the inoculation area of the mice with ethanol and/or iodine solutions. Use lml syringe and a 27- or 30-gauge needle. Mix cells and draw the cells into a syringe without a needle. Inject cells(10×106) subcutaneously (s.c.) or Intraperitoneal injection into the lower flank of the mice or abdominal. The animal tumor assay 1 and 2 should be terminated after 2wks and the assay 3 therapy should start after 1 weeks when the tumors reach an average volume of 0.5cm×0.5cm. Tumor diameters are measured by digital calipers, and the tumor volume is calculated by the formula:Volume= width×2×length×0.52.RESULTS:1 Effect of the treatment on tumorgenicity of Hey cells in miceIn compare with control, The Trichostatin A/Cisplatin treated cells could suppress the tumor size 4-fold and Trichostatin A 0.3um group also has notable suppression effect, the sequence of the tumor suppression is Trichostatin A/Cisplatin>Trichostatin/Cisplatin>Decitabine/Cisplatin>decitabine>control, from weight aspect:Trichostatin A/Cisplatin>Trichostatin >Decitabine/Cisplatin>Decitabine>Cisplatin>control, the tumor growth curve also showed the same effect.2 Effect of the treatment on metastasis of Hey cells in miceWe establised the animal model and selected ten sites as indicator to analysis the effect of the treatment on the metastasis and implantation of ovarain cancer cell Hey in mouse metastasis model. These sites are liver, spleen, omentum, mesenchy, diaphage, stomach, bladder, abdominal wall, pelvic cavity and ovaries, we found that Trichostatin A 0.3um and Cisplatin lum totally suppress the metastasis, and Decitabine, Trichostatain A/Cisplatin and Decitabine/Cisplatin have organ specific patterns.3 Effect of the sequential treatment on the tumor growth inhibitionAccording to the expression of P53, we selected CSPlowdecitabinehigh, DecitabinelowCisplatinlow, CisplatinlowTrichostatinAhigh, TrichostatinAlowCisplatinlow as sequential treatment to look at if these strategies could suppress the growth of tumor, The results indicated that CisplatinlowDecitabinehigh has the most tumor growth inhibition effect.CONCLUSION:Trichostatin A/Cisplatin has the most tumor growth suppression and tumor burden effect and maintain the tumor growth in a low level in a mice tumorgenicity model, Trichostatin A and Cisplatin alone could totally suppress the metastasis in the tumor metastasis model, but this assay need double confirm in the future and try to figure out the detailed mechanism. The sequential treatment of CisplatinlowDecitabinehigh has the most tumor growth inhibition effect, DecitabinelowCisplatinlow also could suppress the growth, it indicated that the combinationof Decitabine and Cisplatin has the synergistic effect.
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