The Study On Biological Characteristics Of Autophagy Induced By Nutrient Depletion And Mechanisms Of Oridonin Effects In Multiple Myeloma

Part I. Autophagy down-regulated by mTOR signaling confers early protection against apoptosis of multiple myeloma cell line exposed to nutrient depletionPurpose:We used nutrient depletion to induce autophagy and apoptosis in MM cell line RPMI8226 cells and observed the relationship between above two.Methods:Four well-recognized markers of autophagy including the MDC fluorescent intensity, the LC3 conversion, the protein and mRNA levels of Beclin 1, and autophagosomal vacuoles assayed by TEM were analyzed. Two standard apoptosis assays including TUNEL and Annexin V/PI staining were used. We used western blot assay to detect the phosphorylation level of S6K1, active caspase 3 protein level, and the translocation level of Bax from cytoplasm to mitochondrial. Statistical analysis was carried out by the Student t-test with p<0.05 representing significance.Results:Our results showed the parameters indicative of autophagy start to increase at 6h, peak at 18h then decline to the baseline at 48h. This process appears to be related to mTOR inhibition. Interestingly, apoptosis starts to increase at 6h, coinciding with the time when autophagy starts, however, the marked increase was observed at 24h, when the autophagic effect declined, then it continuously increases until 48h. We found mitochondria-dependent apoptosis pathway largely plays a role as we observed a clearly relationship between apoptosis and translocation of Bax from cytoplasm to mitochondria as well as activated caspase 3. Furthermore, our analysis of cell death indicated nutrient depletion causes cell death mainly though apoptosis, rather than autophagy. After using 3-MA to inhibit autophagy, we found the inhibition of autophagy facilitates caspase 3 activation and apoptosis before 18h but not at 24h or 48h.Conclusions:Taken together, both autophagy and apoptosis can be induced by nutrient depletion in MM cells. Autophagy occurred in the early phase, appears to inhibit apoptosis. When the stress continued, autophagy declined, and cells underwent apoptosis eventually. Apoptosis is the main pathway of cell death induced by nutrient depletion. mTOR signaling is involved in the regulation of autophagy.Part II The molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in multiple myeloma:a pilot studyPurpose:The part I study has confirmed that nutrient depletion induced autophagy and apoptosis in myeloma multiple. Autophagy has been demonstrated to increase time-dependently at Oh,6h,12h and 18h then decreased at 24h and 48h after nutrient depletion and to be down-regulated by mTORC. Apoptosis has been demonstrated to increase time-dependently and bursted out accompanying with the decrease of autophagy. Also, apoptosis has been demonstrated to be mediated by caspase3 and Bax-dependent mitochondrial apoptosis. The purpose here is to explore the molecular mechanisms underlying the time-dependent autophagy and apoptosis induced by nutrient depletion in multiple myeloma cells.Methods:We studied the transcriptional levels of Deptor, JNK1, JNK2, JNK3, Raf-1, p53, p21 and NFκB1 at Oh,6h,12h,18h,24h and 48h after nutrient depletion in RPMI8226 cells by qRT-PCR and RT-PCR.Results:Raf-1, JNK1, JNK2, NFκB1 and p21 were highly expressed. Deptor and p53 were moderately expressed. JNK3 was lowly expressed. Transcriptional levels of Deptor increased time-dependently at Oh,6h,12h, and 18h then decreased. Its alternation was consistent with autophagy. Transcriptional levels of Raf-1, JNK1, JNK2, p53 and p21 increased time-dependently at Oh,6h,12h,18h,24h and 48h a accompanying with the increase of apoptosis. Transcriptional levels of NFκB1 at 6h, 12h,18h,24h and 48h decreased compared with Oh.Conclusion:All the studied signaling molecules were involved in cellular responding to nutrient depletion in RPMI8226 cells. Deptor mainly contributed to autophagy. Raf-1/JNK/p53/p21 pathway contributed to apoptosis, and NFκB1 inhibited apoptosis. It remains to be studied whether Deptor was involved in both autophagy and apoptosis.PartⅢThe study on mechanisms involved in autophagy and apoptosis induced by oridonin in multiple myelomaPurpose:The purpose is to explore the induction of apoptosis and autophagy, the relationship between them, and involved molecular mechanisms in multiple myeloma cells exposed to oridonin.Methods:RPMI8226 cell vitality was assessed by MTT assay. The morphology of apoptosis and autophagy was observed by TEM. Two apoptosis assays including TUNEL and AnnexinⅤ/PI dual staining were used. The LC3 conversion and localization indicating autophagy level were analyzed by western blot assay and immunofluorescence analysis using QDs605nm-Anti-LC3 fluorescent probe. The intracellular ROS generation was estimated by FCM using the fluorescent probe DCFH-DA. Protein levels of active caspase 3 and Beclin 1 and SIRT1 activity were determined by western blot assay.Results:Oridonin inhibited the proliferation of RPMI8226 cells dose- and time-dependently. Oridonin simultaneously induced caspase 3-dependent apoptosis and Beclin 1-dependent autophagy in a time-dependent manner. Apoptosis was the main pathway of cell death. At the meantime, oridonin resulted in the time-dependent increase of the intracellular ROS generation but decrease of SIRT1 nucleic protein level. Complete inhibition of the intracellular ROS generation by NAC abrogated apoptosis/autophagy induction and SIRT1 nucleic protein level decrease in cells exposed to oridonin. Inhibition of autophagy by 3-MA sensitized oridonin-induced apoptosis accompanied with increase of the intracellular ROS generation but decrease of SIRT1 nucleic protein level.Conclusions:Oridonin can inhibit cell inhibition. Oridonin simultaneously induced apoptosis and autophagy. The intracellular ROS generation mediated apoptosis and autophagy through negative-regulating SIRT1 activity. SIRT1 activity was negative-regulated by intracellular ROS generation(the intracellular ROS generation-SIRT1 axis). The cytoxity of oridonin was mainly executed by apoptosis, but autophagy protected cells against apoptosis acting as a pro-survival mechanism. Autophagy protected against apoptosis involved with this axis.