Experimental Study On The Relationship Between Autophagy And Apoptosis Of Malignant Lymphoma Raji Cells Induced By Arsenic Trioxide And Its Possible Mechanism

Burkitt’s lymphoma originates from follicular germinal center, it is the high-grade malignant B cell lymphoma, and now chemotherapy is still the primary method of treating lymphoma. Many efforts are ongoing to develop innovative and effective therapies, and the most important part of this process is to understand the mechanism of cell death induced by potential chemotherapeutic agents. Arsenic trioxide (AS2O3) has been used successfully in the treatment of patients with newly diagnosed acute promyelocytic leukemia (APL) and other hematological malignancies and solid tumours. Numerous studies have demonstrated that the anti-tumor mechanism of AS2O3 is very complicated, and its deep-seated molecular mechanisms need further study. Autophagy is a tightly regulated lysosomal degradation pathway whereby cells degrade their own damaged organelles and macromolecules to protect cells from environmental stress. Autophagy plays an important role in tumorigenesis and therapy, however, studies onthe interrelation among the different autophagy pathways and the relationship between autophagy and apoptosis have provided conflicting results. A mass studies prompt that autophagy is the protective effect for tumor cells towards chemotherapeutic drugs, thereby promoting cell survival. Yet some researchers believe that hyperactive autophagy will seriously disturb the coordination of cell metabolism, and finally causes the cell death, which is called autophagic cell deathor type Ⅱ programmed cell death, which is different from apoptosis.Recent reports suggest that autophagy and apoptosis often share similar effectors and regulators and a complex cross-talk exists between the two processes. However, the relationship between drug-induced autophagy and apoptosis in tumor cells and their outcomes are still a complete mystery.In this study, the Burkitt’s lymphoma in Raji cells were employed as target to evaluate the relationship between As2O3-inducedautophagy and apoptosis and its molecular mechanisms, and to explore the cross-talks and conversion between macroautophagy (known as autophagy) and chaperone-mediated autophagy (CMA) in cultured Burkitt lymphoma Rajicells when facing serum deprivation and toxic compound As2O3.The results achieved showed as follow:①AS2O3 inhibited the proliferation of Raji cells in dose- and time-dependent manner, and the IC50 of this agent at 24, 48 and 72 h were calculated as (3.51±0.13) μmol/1, (1.57±0.32) μmol/1 and (1.03±0.08) μmol/1, respectively. Raji cells treated with AS2O3 underwent apoptosis and cell cycle arrest. ②AS2O3 could significantly increase the autophagic activity of Raji cells, manifested as promote the formation of autophagic vacuoles and the conversion of soluble LC3-I to lipid bound LC3-Ⅱ, as well as the increased degradation of autophagy substrate P62 protein.3-MA,asan inhibitor of autophagy, could obviously inhibit the autophagic activity, and alleviated the proliferation inhibition, apoptosis and G2/M phase arrest in As2O3-treated Raji cells.Rapamycin, an autophagy revulsant, its roles were exactly opposite to 3-MA. These results provide evidence that the death of Raji cellsinduced by AS2O3 was provided with the shared characteristics of both autophagic and apoptotic pathways.③3-MA could obviously antagonize theup-regulation of Beclin-1 and p53 gene and the down-regulation of Bcl-2 genecaused by As2O3,and the effects of rapamycin were contrary to those of 3-MA. These mean there is a close relationship between apoptosis and autophagy induced by AS2O3 in Raji cells, and the cross-talk may be co-regulated by Beclin-1, p53 and Bcl-2.④ Macroautophagy andCMA are the two best-identified pathways of autophagy. Our results showed that both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first 3-6 hours of nutrition deprivation, and then gradually decreased to near baseline. With nutrient deprivation persisted, CMA progressively increased along with the decline of macroautophagy. On the other hand, in As2O3-treated Raji cells, the macroautophagy activity was also significantly increased, but CMA activity wasn’t rapidly enhanced untill macroautophagy was compulsively inhibited by 3-MA. These suggest that there existsa cross-talk and conversion between macroautophagy and CMA in cultured Burkitt lymphoma Raji cells when facing serum deprivation and toxic compound As2O3.Fundamentally, from the above results, we concluded that the autophagic and apoptotic changes co-existed in the same Raji cells and are regulated by the mutual genes. The strong and lasting macroautophagic activity induced by As2O3 can promote cell death or apoptosis. The environmental stimulation with different intensities will subsequently trigger the different autophagic pathway in tumor cells to meet the responses of stress-caused cell damage. This study may help to better understand the molecular mechanism of anti-lymphoma action of As2O3, and the mutually accommodating answer mode among different autophagic pathways to handle the stress in lymphoma cells, which maylead to a novel idea for the clinical application of regulation of autophagic pathways combining with chemotherapeutic drugs in malignant lymphoma therapy.