Gemcitabine-Induced Autophagy Protects Human Lung Cancer Cells from Apoptotic Death.

PURPOSE: Gemcitabine has been used as a therapeutic drug combined with cisplatin for the treatment of lung cancer patients. However, the prognosis is poor due to acquired resistance. Accumulating studies have revealed that autophagy may contribute to the drug resistance. Therefore, the present study is aimed to clarify the mechanisms underlying gemcitabine-acquired resistance. METHODS: SPC-A1 and A549 cells were incubated with gemcitabine followed by assessment of cell viability with MTT assays. GFP-LC3 transient transfection, MDC staining, and transmission electron microscopy were used to detect the change of autophagy at morphological level. Flow cytometry was used to monitor the effect of 3-MA on gemcitabine-induced apoptosis. Western blot analysis was used to detect the expression of p62, LC3, Beclin-1, ATG5, activated caspase 3, Bax, BNIP3, BNIP3L, and Bcl-2. RESULTS: Our study showed that gemcitabine significantly induced both autophagy and apoptosis in human lung cancer cells SPC-A1 and A549. Of interest was that when autophagy was inhibited by 3-MA, the gemcitabine-induced apoptosis was effectively enhanced, suggesting that gemcitabine can activate autophagy to impair the chemosensitivity of lung cancer cells. Furthermore, the inhibition of autophagy by 3-MA further increased the expression of activated caspase 3, Bax, BNIP3, and BNIP3L, all are critical apoptotic mediators. Contrarily, 3-MA treatment further decreased the expression of Bcl-2, which is an important anti-apoptotic protein. CONCLUSION: Our study indicated that autophagy protected human lung cancer cells from gemcitabine-induced apoptosis, and the combined use of gemcitabine and an autophagic inhibitor in lung cancer patients may be an effective therapeutic strategy.

[Preliminary study on pharmacodynamic evaluation method of Houpo formula particles].

To discuss the feasibility of the pharmacodynamic evaluation method for traditional Chinese medicine (TCM) formula particles, with traditional decoction for reference and the intervention of Magnoliae Officinalis Cortex in rats with ulcerative colitis (UC). First of all, the similarity of traditional Magnoliae Officinalis Cortex decoction and formula particles of different manufacturers was defined by using the IR fingerprint. The UC rat model was established and given Houpo formula particles of different doses and manufacturers, with the decoction for reference, in order to observe disease activity index (DAI), colon mucosa damage index (CMDI), pathologic changes, nitric oxide (NO), endothdin (ET), substance P, vasoactive intestinal peptide (VIP). Their intervention effects on UC rats were compared to study the difference between Sanjiu and Tianjiang Houpo formula particles, in order to demonstrate the feasibility of the pharmacodynamic evaluation method for Houpo formula particles. According to the results, Houpo formula particles showed similar pharmacodynamic actions with the traditional decoction. The pharmacodynamic comparison of Houpo formula particles of different manufacturers showed no statistical significance. The experiment showed that on the basis of the TCM compounds, a prescription dismantlement study was conducted to define target points of various drugs. The traditional decoction was selected for reference in the comparison of corresponding formula particles for their pharmacodynamic equivalence. This method could avoid controversies about single or combined boiling of formula particles, and give objective comments on the pharmacodynamic effect of the formula particles. The method is proved to be feasible.

Decreased expression of Beclin-1 and LC3 in human lung cancer.

Autophagy, a conversed response to stress, has recently been studied in human cancers. Two important autophagic genes-Beclin-1 and LC3 are reported in several human cancers. However, the expressions of Beclin-1 and LC3 in lung cancer have not yet been investigated. In the present study, we investigated the expression of Beclin-1 and LC3, and the relationship between the expression profile and the clinical or pathological changes in human lung cancer. 40 primary lung cancer patients are involved in present study. mRNA expressions of Beclin-1 and LC3-II were detected by Real Time PCR and the protein levels were assessed by immunohistochemistry and western blot. Relative lower expressions of Beclin-1 and LC3-II mRNA were found in the lung cancer tissues compared to counterpart normal tissues. Consistently, the lower amount of Beclin-1 and LC3-II protein was found in lung cancer tissues. However, the expressions of Beclin-1 and LC3-II in lung cancer tissues were not affected by patients' age, gender, smoking, histological type, lymph node metastasis and tumor-node-metastasis (TNM) stage. Both mRNA and protein levels of Beclin-1 and LC3-II were significantly decreased in lung cancer tissues which suggested that autophagy may be involved in the pathogenesis of lung cancer.

Hypoxia-induced autophagy mediates cisplatin resistance in lung cancer cells.

Hypoxia which commonly exists in solid tumors, leads to cancer cells chemoresistance via provoking adaptive responses including autophagy. Therefore, we sought to evaluate the role of autophagy and hypoxia as well as the underlying mechanism in the cisplatin resistance of lung cancer cells. Our study demonstrated that hypoxia significantly protected A549 and SPC-A1 cells from cisplatin-induced cell death in a Hif-1α- and Hif-2α-dependent manner. Moreover, compared with normoxia, cisplatin-induced apoptosis under hypoxia was markedly reduced. However, when autophagy was inhibited by 3-MA or siRNA targeted ATG5, this reduction was effectively attenuated, which means autophagy mediates cisplatin resisitance under hypoxia. In parallel, we showed that hypoxia robustly augmented cisplatin-induced autophagy activation, accompanying by suppressing cisplatin-induced BNIP3 death pathways, which was due to the more efficient autophagic process under hypoxia. Consequently, we proposed that autophagy was a protective mechanism after cisplatin incubation under both normoxia and hypoxia. However, under normoxia, autophagy activation 'was unable to counteract the stress induced by cisplatin, therefore resulting in cell death, whereas under hypoxia, autophagy induction was augmented that solved the cisplatin-induced stress, allowing the cells to survival. In conclusion, augmented induction of autophagy by hypoxia decreased lung cancer cells susceptibility to cisplatin-induced apoptosis.