Screening And Mechanism Research Of Anti-lung Cancer Small Molecule Drugs

Recently lung cancer is the only malignant tumor whose incidence andmortality increased year by year, and is the leading cause of cancer deaths around theworld. It includes small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC), NSCLC accounts for nearly85%of all lung cancer cases and the majorityof them are diagnosed at advanced stages, and are inoperable. At present, thechemotherapy plays a main role in the treatment of advanced lung cancer. Standardplatinum-based chemotherapies provide little improvement in survival with theunsatisfactory results of substantial toxicities. Even with the addition oftarget-therapy, the median survival of metastatic NSCLC patients is about one year.So, anti-cancer drugs of high efficiency and low toxic side effects on the basis oftumor molecular biology, is so urgently needed.Recent studies suggest that apoptosis is a key factor that contributes to theanti-tumor therapies of chemotherapeutic drugs, which has become a focus in theinternational cancer research and the new target for screening of anticancer drugs.Hence the development of novel agents with selectivity against critical apoptotictargets may provide a rational approach to treatment of lung cancer.So far, more than60%of the clinical antitumor chemical drugs are directly orindirectly derived from natural products, many natural compounds have gonethrough long-term natural selection and evolution, they often have reasonablestructure, specific activity and high stability, the research of small molecule drugsand their compounds derived from natural products occupies an important positionin the development of anti-cancer drugs. Many effective components in traditional Chinese medicine have the effect of inducing apoptosis in tumor cells, these activeingredients act on the gene targets of proliferation in malignant tumor cell,selectively inducing apoptosis in tumor cell, without side effects on normal cells,which provides a new research strategy for cancer therapy, in order to provide newhope for improving the prognosis of patients with NSCLC.Dragon’s blood is a kind of traditional Chinese medicine, and is often used totreat injury and bleeding. Dracorhodin perchlorate (DP) is a kind of syntheticanalogues of Dragon’s blood. Reports have found that DP induced apoptosis inhuman cervical cancer cells, human melanoma cells, human breast cancer cells andhuman gastric cancer cells. Chan Su is an important anticancer agent which has beenextensively used to improve the life quality of cancer patients. Cinobufagin (CG) isone of the main compounds of Chan Su, and has significant antitumor effects insome cancer cells, such as liver cancer cells, cervical cancer cells and prostate cancercells. However, little is reported on the anti-tumor potential and the molecularmechanisms of them on human lung cancer. Dehydrocostus lactone (DL) is the maineffective component of traditional Chinese medicine Mu Xiang, and has antitumoractivity in human prostate cancer and leukemia cells. Reports have shown that DLcould induce lung adenocarcinoma cells and large cell cancer cells apoptosis throughER stress, but there was no reports in the effect on human lung squamous carcinomaSK-MES-1cells. Our present study was to investigate the effects of DP and CG inhuman non-small cell lung cancer, and the effect of DL in human lung squamouscarcinoma SK-MES-1cells.In this study, we took four types of NSCLC cell lines for in vitro culture, humanlung squamous carcinoma cells SK-MES-1, human lung adenocarcinoma cellsA549, human large cell cancer cells H460, and human non-small cell lung cancercells H1299. We found that both DP and CG significantly inhibited the growth ofNSCLC cells by MTT assay, we also found that both DP and CG markedly inducedapoptosis in NSCLC cells. The effects of growth inhibition and apoptosis inductionwere in a concentration-response relationship. We used human lung squamous carcinoma SK-MES-1cells as the experimentalmodel to evaluate the potential of DP for further study. The result of flow cytometryshowed that DP induced a G1/G0cell cycle arrest in a dose-dependent manner. Wealso played flow cytometry to detect the mitochondrial membrane potential (MMP)and intracellular reactive oxygen species (ROS) level, and the results showed thatDP significantly decreased the MMP, increased ROS. We played western blot todetect the proteins expression which regulate cell cycle and apoptosis. The resultsshowed that DP markedly increased p53protein expression, and decreased pRbwhich plays an important role in the G1phase transition, which indicates that p53was activated, and its transcription compounds connected with pRb, thus inhibitedpRb activity, blocking the cell cycle at G1phase. We also observed that DPup-regulated the pro-apoptotic protein Bax and down-regulated anti-apoptoticprotein Bcl-2, which means the activation of the mitochondrial pathway, reduced theMMP, promoted the release of apoptotic factors, resulting in the activation ofdownstream caspases and eventually cleavage of their substrate poly ADP-ribosepolymerase (PARP). In addition, we also found a increase of expression of apoptosisinducing factor (AIF), suggests that DP could also induce apoptosis in acaspase-independent way in NSCLC.We used human non-small cell lung cancer H1299cells as the experimentalmodel to evaluate the potential of CG for further study, which has a homozygouspartial deletion of the p53protein and lacks expression of p53protein. The result offlow cytometry showed that CG induced a G2/M cell cycle arrest in adose-dependent manner. We also found that CG dramatically increased the ROSlevel, with the decrease of MMP by flow cytometry, these effects were observablyblocked when the cells were pretreated with N-acetylcysteine (NAC), a specific ROSinhibitor, so was the apoptosis inducing effect, which indicate that CG inducedapoptosis in a ROS dependent manner in H1299cells. We played western blot todetect the proteins expression which regulate apoptosis. Results showed that CGpromoted the up-regulation of Bax and down-regulation of Bcl-2, activated caspase-9and caspase-3, with the proteolytic cleavage of PARP, triggeringchromatin condensation and DNA degradation in order to induce programmed celldeath.We used human lung squamous carcinoma SK-MES-1cells as the experimentalmodel to evaluate the potential of DL. The result of MTT showed that DL couldobviously inhibit the proliferation of lung squamous cancer cells. The result of flowcytometry showed that DL induced a G1/G0cell cycle arrest in a dose-dependentmanner. We also played flow cytometry to detect the apoptosis inducing effect andfound that DL could markedly induce apoptosis in lung squamous carcinomaSK-MES-1cells. We played western blot to detect the proteins expression whichregulate apoptosis. The results showed that DL markedly increased p53proteinexpression, up-regulated the pro-apoptotic protein Bax and down-regulatedanti-apoptotic protein Bcl-2, resulting in the activation of downstream caspase-3andeventually cleavage of its substrate PARP, which suggests that DL could induceapoptosis in SK-MES-1cell via the activation of p53associated mitochondrialpathway.Our study investigated that DP and DL induced apoptosis in SK-MES-1cellsvia activation of p53, while CG induced apoptosis in H1299cells in a ROSdependent manner, both of which further activated mitochondrial pathway. Ourresults reveal that both DP and CG could be potential leading compounds for futuredevelopment of anti-lung cancer therapy.