ABT737Enhances Human Cholangiocarcinoma Cells Sensitivity To Cisplatin Through Mitochondrial Dynamics

Cholangiocarcinoma （CC） is the secondary malignant tumor of thehepatobiliary system. Surgery serves as the main treatment that is only suitable for aminority of patients. CC responds weakly to chemotherapy. Cisplatin is currentlyrecommended as the agent for adjuvant therapy of CC. However, either primaryresistance or acquired resistance comfines cisplatin efficiencyseverely. Thus,understanding the mechanism of the inferior response of CC to cisplatin andidentifying a drug combination based on cisplatin is critical.Mechanisms of thecisplatin resistance in other cancers mainly involve the reduceddrugconcentration,enhanced DNA adducts repair and evading apoptosis.Mitochondria are one of cisplatin intracellular targets. The mitochondrialapoptosis has been considered as one of the main mechanisms for cisplatinanticancer effects.Bcl-2family members can regulate entry into apoptosis mainly bycontrolling MOMP referring to mitochondrial outer membrane permeability.Invarious other cancer cells, Bcl-2family members are related to cisplatinresistanceclosely. There is lack of evidence on whether Bcl-2family membersparticipate in the response of CC to cisplatin.Previous studies indicated that Bcl-2family might influence evading apoptosis of CC induced bycisplatin.Apart form the main role of Bcl-2family in regulating MOMP,they alsoregulate mitochondrial dynamics, which mainly contains mitochondrial fission andfusion as well as mitophagy. Mitochondrial networks are undergoing mitochondrialfusion and fission to generate longer or shorter mitochondria. Most studies suggestthat fission is an important event preceding MOMP, whilefusion can limit apoptosisrelatively. Thus, fusion as a determinant in cisplatin resistance has been proposed inother studies.It is worth noting that mitochondrial fission facilitates the damagedmitochondria degradation, the process referring to mitophagy.Mitophagy is one special form of autophagy.The evidence that chemotherapy （including cisplatin）inductionof mitophagy would protect or damage cancer cells is scarce, whileexcessive mitophagy can induce cell death. Mitochondrial fusion can inhibitmitophagy, therefore may obstruct mitophagic cell death to render cisplatinresistance to CC.Herein, we chose RBE cells as the in vitro model, used the ABT737to illustratethe mechanisms of Bcl-2family and mitochondrial dynamics in the weak responseto cisplatin in human CC, and provided a novel chemotherapeutic strategy andfoundation for CC cells.Methods（1）Cell viability was evaluated by MTT assay. Hoechst33342was used tostain and exhibit apoptotic nuclei. Cells were stained by Annexin V and PI, and theapoptotic cells were quantified by flow cytometry. DCFH-DA was used to markROS, and we observed it by fluorescence microscope. Mitochondrial memberbranepotential was analyzed by flow cytometry following the cells stained by JC-1.（2）We ultilized Western blot to examine the protein level of Bcl-2, Bak andMcl-1in total proteins. We adopted mitochondria isolation kit to acquire theproteins in mitochondrial fraction and cytosolic fraction. The protein level ofcytochrome C or Bcl-2was measured by Western blot.（3）Mitochondrail was marked by MitoTracker Red and observed by laserconfocal microscope, meanwhile nuclei were stained by Hoechst33342to assure therelationship between mitochodnrail fission and fusion and apoptosis. mitochondrialmorphologic proteins, such as Mfn2, OPA1, Drp1and Fis1, were examined byWestern blot. Immunostainning was used to determine the co-location between Drp1and mitochondria, and observed by laser confocal microscope.（4）We utilized Western blot to measure the protein abundance of autophagyassociated proteins p62and LC3. Immunostainning was used to determine theco-location between p62or LC3or acid organelles （marked by LysoTracker Red）and mitochondria （marked by MitoTracker Red or Green）, and observed by laserconfocal microscope.（5）We immunostained p62and stained mitochondria with MitoTracker Red,and observed the intracellular expression of p62and the extent to which it co-localized with mitochondria.（6）According to the sequence of p62（GenBank：NM₀03900）, we usedpGCsilencerTM U6/Neo/GFP/RNAi vector to construct RNAi recombinant plasmidnamed Si-p62. The transfected rate was indicated by the cells percentage lightenedby green fluorescence. And the expression level of p62was measured by Westernblot.（7）After the inhibition of p62expression, MTT assay was selected to evaluatecell viability. Western blot was chosen to detect the expression level of p62, Tom20and Drp1.Results（1）ABT737combined with cisplatin can induce human CC cells to apoptosiseffectively, while the same concentration of cisplatin can seldom cause apoptosis.The combination enhanced the accumulation of ROS, dereased mitochondrialmembrane potential, and promoted the release of cytochrome C from mitochondriato cytoplasm.（2）Compared with the cisplatin treatment alone, ABT737combined withcisplatin can obviously increased the ratio of Mcl-1（32kD）/Mcl-1（40kD） and theprotein level of Bak. The addition of ABT737relieved Bcl-2location onmitochondria cuased by cisplain.（3）Cisplatin alone promote mitochondria of RBE cells fused to each other.Mitochondria presented fragment in the combination group, furthermore, themitochondrial fission occurred prior to apoptosis. Inhibition of mitochondrial fissiondecreased the cytotoxicity of the combination. Meanwhile, downregulated proteinlevel of fusion protein Mfn2as well as OPA1（120kD）, upregulated protein level offisson protein Fis1as well as the increased ratio of Drp1（60kD）/Drp1（80kD） werefound in the combination group. More Drp1（60kD） was promoted to translocate tomitochondria by the combination treatment.（4）Cisplatin or ABT737treatment alone can induce the activation ofautophagy slightly, while ABT737combined with cisplatin can active autophagyseverely. The accumulation of p62or LC3or acid orgenelles co-localized onmitochondria increased obviously and the protein level of Tom20decreased in thecombination group. Silencing p62contributed to the increased Tom20as well as cell viability when cells were challenged by ABT737combined with cisplatin.（5）When cells exposed to ABT737combined with cisplatin, p62aggrationsselectively accumulated more in the cells with fission mitochondria instead of fusedmitochondria. Silencing p62influenced the expression level of Drp1, especially theratio of Drp1（60kD）/Drp1（80kD）.ConclusionHerein, we chose RBE cells as the model in vitro. Firstly, we found that RBEcells responded weakly to cisplatin. While the addition of Bcl-2/Bcl-xL/Bcl-Winhibitor ABT737enhanced the cytotoxicity of cisplatin. Meanwhile thecombination comtributed to the ROS accumulation, decreased mitohcodrialmembrane potential together with increased relase of cytochrome C, whichdemonstrates that sufficient damages on the target organelles （like mitochondria）might be one prerequisite for the anti-tumor efficiency of cisplatin. Furthermore, thecombination disturbed Bcl-2family proteins on both the expression level and thesubcellular location. CC can respond to CDDP by regulation of both Bcl-2familymembers protein abundance and Bcl-2subcellular location.RBE cells responded to cisplatin with mitochondrial fusion, and the addition ofABT737changed the mitochondrial morphology to mitochondrial fission.Mitochondrial fission caused by the combination occurred prior to apoptosis, whileinihibition of fission contributed to increased cell viability. We predict that wepropose that cancer cells may adapt to cisplatin through changes of mitochondrialmorphology, and inhibition of this process might recover the cytotoxic effects ofcisplatin. ABT737can enhance the activation of autophagy resulted from cisplatinand induce obvious mitophagy. The inhibition of mitophagy led to increased cellviability. We speculate that mitophagy participates in the cell death caused by thecombination treatment, and inhibition of mitophagy interrupts the response tocisplatin. Therefore, we support that the resistance of CC to cisplatin is closelyassociated to mitochondrial dynamics.When cells exposed to the combination, p62aggrations selectively accumulatedmore in the cells with fission mitochondria instead of cells with fused mitochondria.Silencing p62influenced mitophagy and the expression of Drp1. That indicates p62may participate in regulating mitochondrial dynamics. In summary, we used RBE cells as the model in vitroand ABT737as theinhibitor to interrupt the balance within Bcl-2family members. We demonstratedthat CC might respond to cisplatin by influencing Bcl-2family proteins, limiting themitochondrial damage and regulating mitochondrial dynamics.ABT737can affectall the processes mentioned above, therefore enhance the sensitivity to cisplatin ofCC. ABT737combined with csiplatin might provide a novel chemotherapeuticstrategy for CC.