Effect of Lidamycin on Telomerase Activity in Human Hepatoma BEL-7402 Cells

Objective To investigate the effect of lidamycin （LDM） on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. Methods Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-13-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. Results Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-13-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased （P〈0.01） in LDM-treated hepatoma BEL-7402 cells. Conclusion Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addition,enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer.Furthermore,the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer.In this study,a novel FR-directed,macropinocytosis-enhanced,and highly cytotoxic bioconjugate folate(F)-human serum albumin(HSA)-apoprotein of lidamycin(LDP)-active enediyne(AE)derived from lidamycin was designed and prepared.F-HSA-LDP-AE consisted of four moieties:F,HSA,LDP,and AE.F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells.Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells.By in vivo optical imaging,F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice,showing clear and lasting tumor localization for 360 h.In the MTT assay,F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines.It also induced apoptosis and caused G2/M cell cycle arrest.F-HSALDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice.At well-tolerated doses of 0.5 and 1 mg/kg,(i.v.,twice),the inhibition rates were 91.2%and 94.8%,respectively(P<0.01).The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Excellent effects and possible mechanisms of action of a new antibody–drug conjugate against EGFR-positive triple-negative breast cancer

Background:Triple-negative breast cancer(TNBC)is the most aggressive subtype and occurs in approximately 15%–20%of diagnosed breast cancers.TNBC is characterized by its highly metastatic and recurrent features,as well as a lack of specific targets and targeted therapeutics.Epidermal growth factor receptor(EGFR)is highly expressed in a variety of tumors,especially in TNBC.LR004-VC-MMAE is a new EGFR-targeting antibody–drug conjugate produced by our laboratory.This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action.Methods:LR004-VC-MMAE was prepared by coupling a cytotoxic payload(MMAE)to an anti-EGFR antibody(LR004)via a linker,and the drug-to-antibody ratio(DAR)was analyzed by HIC-HPLC.The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset(GSE41313)and Western blotting.MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE(0,0.0066,0.066,0.66,6.6 nmol/L),and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation.The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VCMMAE concentrations(2.5 and 5 nmol/L)with wound healing and Transwell invasion assays.Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells(MDA-MB-468 and MDA-MB-231 cells).The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo.Briefly,BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDAMB-231 cells.Then they were randomly divided into 4 groups(n=6 per group)and treated with PBS,naked LR004(10 mg/kg),LR004-VC-MMAE(10 mg/kg),or doxorubicin,respectively.Tumor sizes and the body weights of mice were measured every 4 d.The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry.Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR,ERK,MEK phosphorylation and tumor stemness marker gene expression.Results:LR004-VC-MMAE with a DAR of 4.02 were obtained.The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells(P<0.01).LR004-VC-MMAE inhibited the proliferation of EGFRpositive TNBC cells,and the ICvalues of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were(0.13±0.02)nmol/L and(0.66±0.06)nmol/L,respectively,which were significantly lower than that of cells treated with MMAE[(3.20±0.60)nmol/L,P<0.01,and(6.60±0.50)nmol/L,P<0.001].LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells.Moreover,LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability.In TNBC xenograft models,LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36.Surprisingly,tumor recurrence was not observed until the end of the experiment on day 52.In a mechanistic study,we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G/M phase in MDAMB-468[(34±5)%vs.(12±2)%,P<0.001]and MDA-MB-231[(27±4)%vs.(18±3)%,P<0.01]cells.LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4,Sox2,KLF4 and EpCAM.Conclusions:LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the expression of cancer stemness marker genes.It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.