The Anti-tumor Effect And Underlying Mechanism Of6r:Both In Vitro And In Vivo Studies

BackgroundsEthacrynic acid (EA) is a glutathione S-transferase P1-1(GSTπ) inhibitor with weak anti-proliferative ability in tumor cells. But its low efficacy and high side effects have limited its pharmacological development as an effective chemotherapeutic agent. We designed and synthesized a series of EA oxadiazole derivatives using the principle of bioisosterism. We found that the compound5-[2,3-Dichloro-4-(2-methylene-l-ox obutyl) phenoxymethyl]-3-methyl-1,2,4-oxadiazole (6r) possesses strongly anti-tumor effect against a series of cancer lines. Thus, we evaluated the efficacy of6r as a candidate agent for cancer treatment.MethodsIn vitro assay:MTT assay was performed to evaluate the anti-tumor effect of6r on a series of cancer cells. We chose prostate cancer cells PC3and colon cancer cells SW620for further studies. Hoechst33258staining analysis and FITC-Annexin V/PI double staining assay were carried out to examine the apoptosis effect of cancer cells. PI staining assay was performed to measure the cell cycle arrest of cancer cells after6r treatment. Western blotting analysis was employed to examine the expression of GSTπ, EGFR/PI3K/Akt and cyclin Dl in cancer cells.In vivo assay:The in vivo efficacy of6r was assessed in nude mice bearing SW620subcutaneous xenografts, PC3subcutaneous xenografts and PC-3M-Luc-C6orthotopic xenografts. Mice were randomly into several groups, one group as control to receive vehicle, other groups were treated with6r,5-Fluorouracil (5-Fu) or Mitoxantrone (MA) at indicated dosage by intravenous injection, nude mice were treated every two days for several consecutive weeks. At the end of the experiments, mice were sacrificed and the tumors were moved and weighted. Effect of6r on tumor growth was expressed as a percentage to tumor weight in the control group. In addition, western blotting assay was performed to examine the expression of GST π, apoptosis-related proteins and EGFR/PI3K/Akt in tumor tissues.ResultsIn vitro results:The effect of6r on cell proliferation was evaluated using the MTT assay against a panel of10human tumor cell lines.6r significantly inhibited the proliferation of PC3and SW620cells with IC50value of3.79μM and4.89μM. We chose SW620and PC3cells, which6r showed to have the most effect, for the following experiment. Hoechst33258staining results showed that2μM,4μM6r induced the nuclear chromatin, condensation, or in blocks fragmentation change role, or gathering around to the nuclear membrane both in PC3and SW620cells. FITC-Annexin V/PI double staining showed that the apoptosis rate was28.8%and29.41%respectively after treatment with4μM6r for24h in SW620cells and PC3cells. PI staining results showed that6r can induce cancer cell cycle arrest at S phase. The percentages of cell in S-phase were66.85%in SW620cells and22.79%in PC3cells respectively after6r treatment. Western blotting results showed that6r has no effect on the expression of GSTπ,6r can inhibit the expression of EGFR/PI3K/Akt as well as Cyclin D1in PC3cells.In vivo results:6r significantly delayed the growth of SW620subcutaneous xenografts, PC3subcutaneous xenografts and PC-3M-Luc-C6orthotopic xenografts in nude mice. Treatment of6r at8mg/kg strongly delayed the growth of tumors by44.17%in SW620xenografts after three consecutive weeks; by63.3%in PC3xenografts after three consecutive weeks and60.5%in PC-3M xenografts after four consecutive weeks without significant toxicity. Therefore,6r possesses strong anti-tumor effect in vivo. Western blotting resulted that6r has no effect on the expression of GSTπ in SW620xenografts.6r could significantly increase the expression of cleaved-caspase-3, promote the release of Cytochrome C into cytoplasm, and decline the radio of bcl-2/Bax in tumor tissues.6r could also inhibit the expression of EGFR/PI3K/Akt in PC-3M xenografts.Conclusion6r possesses strong anti-tumor effect against SW620and PC3cells both in vitro and in vivo. Further study showed that the anti-tumor effect of6r was associated with inhibiting EGFR and PI3K/Akt signaling pathway and inducing cancer cell apoptosis via bcl-2/Bax-Cyt-C-caspase-3mitochondrial signaling pathway.6r could also induce cell cycle arrest at S phase via inhibition of Cyclin D1in cancer cells. Therefore,6r was a promising anti-cancer agent for cancer treatment.