Studies On The Molecular Mechanisms Of Lidamycin Alone And In Combination With Gefitinib Against NSCLC

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer patients. In spite of new treatments, the overall five-year survival rate remains about 14% and most patients present with advanced disease. Treatment outcomes for NSCLC patients still are considered disappointing because of chemo-resistance and dose-accumulated toxicity.LDM showed extremely potent cytotoxicity toward culture cancer cells and markedly inhibited the growth of transplantable tumors in mice and human cancer xenografts in nude mice. The potent efficacy of LDM was ascribing to its DNA strand-scission activity. Recent study displayed the chromosomal aberrations and telomere dysfunction induced by LDM. LDM is currently being evaluated in phase II clinical trials as a potential chemotherapeutic agent in China.Studies on the molecular mechanisms of LDM alone and in combination with gefitinib against NSCLC were investigated.1. Effects of LDM on NSCLC cells in vitro1.1. Antiproliferative activities were measured by MTT assays. Remarkable growth inhibition effects of LDM were found in all tested NSCLC cell lines. The IC50 values of LDM for all the cell lines tested were much lower than those of the other chemotherapeutic drugs.1.2. Flow cytometry combined with FITC-Annexin V/PI staining showed that LDM induced apoptosis of NSCLC cells dose-dependently. H460 cell line was the greatest sensitive cells which consisted of 43.30%±4.26% ( P<0.001) apoptotic cells after exposure to 0.5 nM LDM and the apoptotic cells induced by 2nM LDM reached 69.87±3.29% (P<0.001). LDM also induced significant apoptosis in H157 and A549 cells, but the extent of apoptosis was lower than that of H460 cells.The Hoechst 33342 staining was used to assess the change in nuclear morphology after the treatment of LDM. The nuclei of untreated cells and low concentration LDM treated cells were normal and exhibited diffused staining of the chromatin. After exposure to LDM for 48 h, most cells of the three cell lines treated with 1nM and 5nM LDM presented typical morphological changes of apoptosis such as chromatin condensation, nucleus shrink and the formation of apoptotic bodies.The ratios of apoptosis cells detected with TUNEL method were increased with the concentration of LDM increasing in H460 cells and H157 cells.1.3. The flow cytometric cell cycle analysis showed that LDM at the doses of below 1 nM induced G₂/M arrest, 1nM and 2nM LDM produced an S-phase block in addition to G2/M arrest.Sub 2N DNA formation was assessed for measuring apoptosis quantitatively. The peak effect was in dose dependent and achieve to the maximum percentage of 52.55±4.45 of apoptotic cells in H460 cells exposed to 2 nM of LDM. At 1 nM and 2 nM, LDM also induced significant apoptosis in H157 and A549 cells, but the extent of apoptosis was lower than that of H460 cells.1.4. Western blotting showed that LDM significantly increased caspase-3 and caspase-7 activities as well as PARP cleavage. The decreased Bcl-2 and NF-κB indicated that mitochondria-caspase cascade was responsible for LDM-induced apoptosis. The decreased CyclinB1 and the up-regulated of P53 and P21 were the potent evidence for G₂/M phase arrest.1.5. The effects of LDM on the migration and invasion of the NSCLC cells were examined with transwell chamber assay. A dose dependent reduction in migration or invasion of the three cell lines was found after exposure to various concentrations of LDM. Both of H157 and A549 cells have high invasion activities; however, H460 cells have the weakest migration and invasive activity. 1.6. Zymography analysis showed that LDM strongly inhibited the secretion of MMP-9 in all the tested cells and the secretion and activation of MMP-2 in HT-1080 cells and H157 cells dose dependently. However, the MMP-2 levels in H460 cells and A549 cells were little affected by LDM.1.7. Western blot analysis showed that LDM downregulated the levels of KDR, VEGF, COX-2, MMP-9 and MMP-2 dose dependently with different extent in the three cell lines.1.8. Western blot analysis showed that LDM exhibited potent effects of antiproliferation, apoptosis induction, cell cycle arrest and anti-invasion by regulating the activities of EGFR signaling pathway targets, although to the various extents in different cell lines. The most significant coincidence was the activation of RAF/MEK/ERK signal pathway in the three cell lines.1.9. Western blot analysis and FITC-Annexin V/PI staining showed that the up-regulations of p-ERK1/2 levels in the three cells treated with LDM were ablated by U0126 (MEK1/2 inhibitor). Meanwhile, U0126 attenuated apoptosis induced by LDM. The results indicated a correlation between LDM-mediated apoptosis and ERK activation.2. In vivo antitumor activity of LDM on A549 xenograftsTreatment with LDM at the doses of 0.02 mg/kg and 0.04 mg/kg inhibited the growth of human adenocarcinoma A549 xenografts by 39.5% and 57.6%, respectively. The body weights of animals showed no significant differences between control and treated groups.3. Effects on H460 cells and A431 cells by the combination of LDM with gefitinib3.1. MTT assay showed that LDM was much more potent than gefitinib since the IC₅₀ values of LDM for cell lines tested were lower than those of gefitinib. The levels of EGFR were associated with the sensitivity to gefitinib, since potent inhibition (IC₅₀: 0.28±0.03μM) was observed in highly EGFR-expressing A431 cell line whereas the moderate EGFR-expressing NSCLC cells were relatively resistant (16.04±2.96μM～19.57±6.6μM).3.2. To determine whether a potentiation of the antiproliferative activity could be obtained by the combination of LDM and gefitinib, a series of experiments were performed on H460 and A431 cell lines treated with different doses of each drug. Slight synergistic growth inhibitory effects at most combinations (CDK1) were found in the two cell lines. The CDI was<0.70 at the combination dose [gefitinib (μM)/LDM (nM):5/0.1] in H460 cells, [gefitinib (μM)/LDM (nM):0.1/0.01] in A431 cells. The synergistic effects were achieved at lower gefitinib concentration in A431 cells than in H460 cells.3.3. Induction of apoptosis by LDM and gefitinib in H460 and A431 cell lines were measured by flow cytometry combined with FITC-Annexin V/PI staining. The results showed that the percentage of Annexin V-positive cells increased dose dependently in the two cell lines after treated by the two drugs. LDM showed similar potent apoptosis induction effects on H460 cells and A431 cells. However, A431 cell line was more sensitive than H460 cell line to gefitinib; there occurred 30.70%±1.69% ( P<0.01) apoptotic cells as A431 cells exposed to 1μM gefitinib whereas only 13.60±1.37% (P<0.05) apoptotic cells induced by 20μM gefitinib in H460 cells.3.4. FITC-Annexin V/PI analysis showed that the combination treatment induced more intensive apoptosis when compared to that by each agent alone in A431 cells. The lower dose combination, 0.1μM gefitinib plus 0.01 nM LDM, induced apoptosis in 31.87% of cells, whereas the same doses of gefitinib and LDM given alone resulted in apoptosis in only 12.79% and 6.71% of cells, respectively. Likewise, at the higher dose level (1μM gefitinib plus 0.1 nM LDM) the apoptotic rate was 53.93%, clearly superior to the 31.89% and 21.45% of apoptosis with single-agent gefitinib and LDM, respectively. The percentage of apoptotic cells was much lower in H460 cells even in higher dose of gefitinib. However, LDM could slightly potentiate the apoptotic effect of gefitinib at certain doses. 20μM gefitinib plus 0.5 nM LDM induced apoptosis in 53.85% of cells, whereas the same dose of gefitinib and LDM given alone resulted in only 12.63% and 42.88% of apoptotic cells, respectively.3.5. Western blot analysis showed that a dose-dependent increase in PARP cleavage was observed in A431 cells exposed to gefitinib. More significant increase in PARP cleavage was observed after combined treatment. However, gefitinib barely increase the PARP cleavage in H460 cells at the doses tested. 10μM gefitinib plus 0.5 nM LDM introduced slight increase in PARP cleavage. Furthermore, greater down-regulating of the anti-apoptotic molecule NF-κB in the two cell lines also related to enhancement of apoptosis by combined treatment.3.6. Western blot analysis evaluated the effects of gefitinib and LDM on EGFR signaling pathway molecules in H460 and A431 cells. Phosphorylation of EGFR, ERK and Akt decreased in gefitinib-treated A431 cells and a more pronounced decrease in the levels of phosphorylation of each marker was observed after the combined treatment. 10μM gefitinib plus 0.5 nM LDM slightly decreased the level of p-EGFR but not of p-ERK and p-Akt as compared to each single agent in H460 cells. Pretreatment with gefitinib inhibited EGF-induced phosphorylation of EGFR and Akt in H460 cells. Combined treatment markedly inhibited the increment of EGFR and Akt phsphorylation induced by EGF. However, the inhibition of the activation of ERK by combination was similar to that of gefitinib alone.4. Comparison in vivo antitumor activity of LDM and gefitinib on H460 xenograftsTreatment with LDM at the dose of 0.025 mg/kg and 0.05 mg/kg inhibited the growth of human large cell lung carcinoma H460 xenografts by 52.8% and 72.4%, respectively. Oral administration of gefitinib at the dose of 50 mg/kg inhibited the growth of H460 xenografts by 69.4%. The inhibitory effect was comparable to the tolerable dose of LDM of 0.05 mg/kg. The body weights of the animals showed no significant differences between the control and treated groups.