Potency Of Lidamycin On Ovaraian Carcinoma Suppression And Hsp90 Inhibitors Geldanamycin And Its Analogue Enhance The Cytotoxicity Of Lidamycin

Potency of Lidamycin on ovaraian carcinoma suppression and Hsp90 inhibitors Geldanamycin and its analogue CNDG enhance the cytotoxicity of lidamycinTumor resistance to DNA damaging agents is often associated with enhanced DNA repair activity. Reducing DNA damage repair is proved to be an effective way to sensitize cancer cells to DNA damaging agents. Enediyne lidamycin (LDM, also called C-1027) showed extremely potent cytotoxicity by inducing DNA damage and Hsp90 is an interesting target for anticancer treatment recently. In this research we investigated whether Hsp90 inhibitors, geldanamycin (GDM) and its analogue CNDG, could further enhance the potent cytotoxicity of LDM. The survival of cells after DNA double-strand breaks (DSBs) is heavily dependent on the DNA damage repair. Phosphatidylinositol-3 kinase-related kinases (PIKKs) members such as ATM and ATR play pivotal roles in response to DNA damage and maintaining genome integrity. They transduce genomic stress signals to halt cell cycle progression and promote DNA repair. After DNA damaging agent treatment PIKKs is activated to attempt the cell cycle arrest and DNA damage repair. ATM responds mainly to DSBs, whereas ATR is activated by single stranded DNA and stalled DNA replication forks. Further, the activation of ATM and ATR triggers G2/M phase arrest and some DNA repair enzymes. Chk2 can be directly activated by ATM and Chkl by ATR. Both Chkl and Chk2 can arrest cell cycle at G2/M phase, retarding DNA replication and stimulating the repair of damaged DNA.Lidamycin (LDM) is an enediyne antitumor antibiotic produced by Streptomyces globisporus C-1027. It is currently being evaluated in phaseⅡclinical trials as a potential chemotherapeutic agent in China. LDM induces DSBs leading potent cytotoxicity and marked inhibition of tumor growth in vivo. As a DNA damaging agent LDM induced DNA damage repair and this might decrease the effect of LDM.Hsp90 is important for stabilization and trafficking of tyrosine and serine/threonine kinases that are activated in response to genotoxic stress, including those that are essential for survival of cancer cells. GDM is an ansamycin antibiotic that inhibits HSP90 by binding to the NH2-terminal ATP binding domain, leading to degradation of HSP90 clients. Some Hsp90 inhibitors are in phaseⅠ/Ⅱclinical trials in combination with radiation or other chemotherapeutic agents. Many reports show that Hsp90 inhibitors enhance the effect of DNA damaging agents, and some of them indicate that Hsp90 inhibitor decreases DNA damage repair through inhibiting ATM dependent DNA damage repair.In this report Hsp90 inhibitors GDM and its analogue CNDG were found to enhance the anticancer effect of LDM. Our results suggested that GDM pretreatment potentiated the cytotoxity of LDM by decreasing the LDM-induced DNA damage repair. To our knowledge, it is the first report about Hsp90 inhibitor enhancing potent cytotoxicity of LDM or enediyne antitumor antibiotic.1. LDM effects on SKOV-3 cellsBy MTT assay, LDM inhibited the proliferation of SKOV-3 cells. Examined with Annexin V-FITC/PI double staining flow cytometry, the apoptotic rates in 0.01 nM and 0.05 nM LDM treated cells were 10.92% and 44.78% respectively; at 0.05 nM, LDM dramatically increased the comet tail; and 0.1 nM of LDM inhibited the phosphorylation of HER2. In the hypoxia condition, LDM reduced the level of HIF-1α.2. GDM and its analogue sensitize SKOV-3, MCF-7 and Bel-7402 cells to LDMIt has been demonstrated that inhibition of Hsp90 could potentiate the efficacy of ionization radiation, antimetabolites and alkylating agents. So we investigated whether inhibition of Hsp90 could potentiate the cytotoxicity of LDM. MTT assays were used on different cancer cells. Cells were exposed to LDM for 24 h after 16 h preincubated with GDM. Under our experimental conditions, low concentrations of LDM (0.01 and 0.05 nM) did not show extensive growth inhibition of SKOV-3 cells. In the following experiments, we chose 0.01 and 0.05 nM LDM for SKOV-3 as appropriate doses to detect the potentiation effects of Hsp90 inhibitor.By MTT assay, LDM markedly inhibited cell growth by inhibiting Hsp90. Combined with 100 and 500 nM GDM,0.01 and 0.05 nM LDM induced more growth inhibition than LDM alone. We compared the cytotoxicity of the GDM-LDM combination to the effect of the two agents alone using the median effect method, which determines whether the cytotoxicity for the combination is greater than (CI＜1), equal to (CI=1), or less than (CI>1) the additive effect of the individual agents. Under the indicated doses, most of the CI values were less than 1 and some of them were less than 0.7. These results suggested that the combination of LDM and GDM showed a synergistic effect in SKOV-3 cells. Notably,17-(6-cinnamamido-hexylamino)-17-demethoxygel- danamycin (CNDG), a derivate of GDM synthesized in our laboratory had similar protentiation effect on LDM cytotoxicity to cancer cells. Under the same condition, we also chose Bel-7402, A549 and MCF-7 cells to check this combination method, and the results were quite similar.3. GDMpotentiats the apoptosis-inducing effect of LDMWestern blot analysis was used to detect the cleavage of PARP, an indicator of caspase-mediated apoptosis. The 89-kDa cleaved fragment of PARP increased dose-dependently in SKOV-3 cells exposed to LDM. Enhanced levels of cleaved PARP appeared after combination treatment.To confirm the augmentation of LDM-induced apoptosis by GDM, we performed annexin V/PI staining assay. Consistent with above results, LDM in combination with GDM pretreatment induced more apoptosis than LDM alone. The apoptosis cell ratios were increasing from 14% to 32.2%.4. GDM enhances LDM-induced DNA damageTo examine the effects on DNA damage induced by LDM in the absence and presence of GDM, the phosphorylation of Ser-139 of histone H2AX was compared by immunoflurorescence and Western blot. The treatment of LDM or GDM alone did not increase yH2AX protein, however, the combination of GDM and LDM markedly enhanced yH2AX levels. The figure of yH2AX foci by immunoflurorescence also confirmed the result.The alkaline comet assay could provide a direct measure of DSBs. There was no significant DNA damage in SKOV-3 cells exposed to either LDM or GDM alone. Significantly more DNA damage in combination-treated cells versus either LDM or GDM alone treated cells.5. GDM inhibits LDM-induced DNA damage repairyH2AX levels after were determined 1 h and 6 h LDM treatment. LDM increased the number of yH2AX positive cells. However, the number of yH2AX positive cells was reduced dramatically after 6 h treatment as compared with 1 h treatment, indicating that damaged DNA was partially repaired. The combination of GDM and LDM did not seriously affect the yH2AX positive cell number compared with LDM alone at 1 h treatment. Notably, after 6 h treatment, the percentage of yH2AX positive cells was significantly increased in the combination-treated cells as compared with LDM alone. Time course analysis confirmed that GDM reduced DNA damage repair in LDM-treated cells.6. GDM abrogates LDM-induced G2/M arrest and phosphorylations of Chkl and Chk2Most of DNA damage agents including LDM induce G2/M phase arrest. Treated by 0.05 nM LDM cells at G2/M phase were increased from 15.28%(control) to 76.79%. At the same condition,200 nM GDM did not induce G2/M arrest significantly. However, the pretreatment of GDM down-regulated the number of G2/M phase cells, and the percent of G2/M phase in LDM-treated cells was decreased from 76.79% to 29.33%. By Western blot analysis the phosphorylations of Chkl and Chk2 induced by LDM were decreased by pretreatment of GDM.7. GDM compromised LDM-induced ATM activity, the protein level of ATRIP and DNA damage related proteinsTo address the molecular mechanism involved in GDM mediated inhibition of DSB repair, we focused on LDM-induced ATM and ATR activity. As shown, ATM phosphorylation was increased within 1 h of LDM treatment. By contrast, cells pretreated with GDM had a decrease of phospho-ATM in LDM treated cells. These suggest that GDM interferes with the activation of ATM-mediated DSB repair in response to LDM.ATR is also an important kinase for LDM induced DNA damamge response. Previous studies showed that both ATR and ATRIP played an important role in this pathway, and ATRIP could regulate ATR levels. Thus, we checked ATR and ATRIP level of cells treated with GDM. The results showed that ATRIP dramatically decreased after 16 h GDM-treatment, while ATR had no change. We then examined the states of ATM, ATR and ATRIP in SKOV-3 cells treated with 200 nM GDM for 40 h,0.05 nM LDM for 24 h or with combination, that is, LDM was added after 16 h of GDM pretreatment. The total proteins of ATM and ATR remained unchanged after 16 h GDM. After 40 h GDM pretreatment, the total levels of ATM and ATR were decreased observablely.Then the influence of GDM on ATRIP expression was examined for cells in the presence or absence of proteasome inhibitors. For this study, SKOV-3 cells were incubated for 16 h with 200 nM GDM along with 10μM proteasome inhibitor MG132. The cells were lysed and analyzed for ATRIP protein levels. Evidently, incubation of cells with MG132 abolished the decrease of ATRIP protein in SKOV-3 cells induced by GDM treatment. Also, GDM could decrease other proteins that are related with DNA damage response.In summary, we found that Hsp90 inhibitors could enhance the potent cytotoxicity of LDM. The mechanism was dependent on, at least in part, increasing DNA damage and depressing cell response to DNA damage. Finally, our data suggests that the combination strategy of Hsp90 inhibitors and LDM may represent a novel and promising approach for ovarian cancer treatment.