Calcium Carbonate Nanocarriers Enhance Sensitivity Of Hepatocellular Carcinoma To Cisplatin Via Disruption Of Mitochondrial Turnover Pathways

Background:Hepatocellular carcinoma(HCC)has a poor prognosis,with its mortality ranking second among tumors in China.Surgical intervention stands as the primary curative treatment for HCC;however,the 5-year recurrence rate post-surgery reaches as high as 70%.This high recurrence rate is mainly attributed to the lack of postoperative adjuvant therapy to prevent HCC recurrence.Currently,clinical research on targeted therapy and immunotherapy for adjuvant treatment of HCC post-surgery is gradually advancing.Nevertheless,due to the heterogeneity of HCC,genomic sequencing reveals that only 25%of HCC tumors possess a potential target.Effective targeted or immune adjuvant treatment strategies for postoperative HCC have yet to be discovered.Traditional cytotoxic drugs can broadly act on heterogeneous cells of HCC without specific target requirements,hence presenting high application potential in adjuvant therapy postsurgery for HCC.In China,guidelines recommend Transcatheter Arterial Chemoembolization(TACE)using cisplatin as the standardized chemotherapy drug for localized postoperative chemotherapy,indicating that the relatively high concentration of cisplatin in liver cancer tissue can yield favorable therapeutic effects.Platinum-based drugs have significant application value and prospects in HCC treatment.Thus,enhancing the sensitivity of HCC to chemotherapy is of great significance for systemic chemotherapy and postoperative adjuvant therapy.Traditionally,cisplatin is believed to act on the nucleus DNA of cells.However,an increasing body of research indicates that cisplatin can exert its cytotoxic effects through organelles such as mitochondria and lysosomes.Among these organelles,mitochondria play a crucial role in regulating cell death pathways such as apoptosis,necrosis,and pyroptosis,thereby determining the "life and death" of cells.Notably,the high affinity and damage caused by cisplatin to mitochondrial DNA(mtDNA)make mitochondria another important target of cisplatin action.However,when mitochondria are damaged,mitochondrial quality control mechanisms are activated to repair and eliminate damaged mitochondria,thereby maintaining cellular homeostasis.Increasing levels of mitochondrial damage will sequentially activate mitochondrial quality control at the molecular,organelle,and cellular levels.Previous studies by our research group have found that under the action of cisplatin,HCC cells can initiate organelle-level mitochondrial quality control,namely mitophagy,to resist the cytotoxic effects of cisplatin.Excessive mitochondrial damage further triggers cellular-level mitochondrial quality control,namely mitochondrial apoptosis.Mitochondrial quality control is regulated by two synergistic mechanisms:the clearance of damaged mitochondria and the synthesis of healthy mitochondria,namely mitophagy and mitochondrial biogenesis,which together constitute mitochondrial turnover.To maintain proper mitochondrial homeostasis,the process of clearing damaged mitochondria must be strictly regulated and balanced by mitochondrial biogenesis.Mitochondrial biogenesis,as a multi-step process,includes mtDNA transcription,translation,and nuclear-derived transcript translation,mediated by PGC1α and its downstream regulatory factors NRF1/2 and TFAM.Therefore,we speculate that the mitochondrial turnover pathway plays a role in cisplatin resistance in HCC.In response to the involvement of mitochondrial turnover in cisplatin resistance in HCC,this study designed and prepared a carbonate calcium nanoparticle carrier loaded with cisplatin and siPGC-1α,namely CDDP-ICG@CaCO3/PAA+siPGC-1α.On one hand,it releases CDDP and Ca2+in HCC cells,causing mitochondrial Ca2+overload,exacerbating mitochondrial damage,and inducing cell apoptosis.On the other hand,siPGC-1α can further block mitochondrial biogenesis,interfering with multiple dimensions of the mitochondrial turnover pathway in HCC cells,thereby enhancing HCC sensitivity to cisplatin.Methods:(1)Perform MTT assay to assess the cell viability of Huh7 and HCCLM3 cells after 48 hours of treatment with CDDP;(2)Utilize Western Blot to examine the expression changes of mitochondrial autophagy-related proteins PINK1,Parkin,LC3,and p62 after treatment with CDDP.Conduct MTT assay to evaluate changes in cell viability following treatment with different concentrations of CDDP or in combination with the mitochondrial autophagy inhibitor Mdivi-1 for 48 hours.Analyze changes in apoptosis levels of HCC cells using flow cytometry after inhibiting mitochondrial autophagy and treating with CDDP for 48 hours;(3)Conduct RT-qPCR experiments to measure the changes in relative mtDNA copy numbers in Huh7 and HCCLM3 cells treated with CDDP for different durations.Use Western Blot to assess the expression changes of mitochondrial biogenesis-related proteins PGC-1α,NRF2,TFAM,and mitochondrial-related protein TOM20 after treatment with CDDP.Employ MTT assay to analyze changes in cell viability of HCC cells after PGC-1α knockdown and treatment with different concentrations of CDDP.Use flow cytometry to investigate changes in apoptosis levels of HCC cells after PGC1α knockdown and treatment with CDDP;(4)Synthesize calcium carbonate nano-carriers using a one-pot method and characterize them,including morphology and particle size using TEM,crystal structure analysis using XRD,absorbance spectra using UV-Vis,component changes with temperature using thermal analysis,Zeta potential and DLS analysis using dynamic light scattering,CDDP content determination using ICP,photothermal conversion efficiency after 808nm laser irradiation using infrared thermography,CDDP release under different pH and light conditions using dialysis method and ICP,siPGC-1αloading efficiency using agarose gel electrophoresis,particle size analysis of different concentrations of BSA at different times using a particle size analyzer,and cellular uptake of CDDP-ICG@CaCO3/PAA+siPGC-1α using flow cytometry;(5)MTT assay was used to evaluate the safety of CaCO3/PAA and the impact of CDDP-ICG@CaCO3/PAA+siPGC-1α on cell viability.Analyze changes in apoptosis levels of cells using flow cytometry after treatment with CDDPICG@CaCO3/PAA+siPGC-1α.Investigate changes in expression levels of apoptosisrelated proteins in HCC cells at the cellular level after treatment with CDDPICG@CaCO3/PAA+siPGC-1α using Western Blot;(6)Western Blot was used to detect the expression changes of mitophagy pathwayrelated proteins PINK1,Parkin,LC3,and p62 proteins after the action of CDDPICG@CaCO3/PAA+siPGC-1α,and MTT experiment was used to detect CDDPICG@CaCO3/PAA+siPGC-1α.Changes in cell viability after 48 hours of treatment with or combined with the mitophagy inhibitor Mdivi-1;(7)RT-qPCR experiment was used to detect changes in relative mtDNA copy number of CDDP-ICG@CaCO3/PAA+siPGC-1α treated at different times,and Western Blot was used to detect mitochondrial biogenesis-related proteins PGC-1α,NRF2,TFAM and mitochondria-related proteins.Expression changes of TOM20;(8)Rhod-2 AM was used for staining,and flow cytometry was used to detect the relative mitochondrial Ca2+level of CDDP-ICG@CaCO3/PAA+siPGC-1α.The mitochondrial Ca2+content was measured using a mitochondrial extraction kit and ICP technology.Conduct detection and MTT experiment to detect the effect of CDDPICG@CaCO3/PAA+siPGC-1α or combined calcium ion chelator BAPTA on cell viability;(9)Mito-SOX staining was used to detect changes in mtROS levels under the action of CDDP-ICG@CaCO3/PAA+siPGC-1α by flow cytometry,and JC-1 staining was used to detect CDDP-ICG@CaCO3/PAA+siPGC-1α.Changes in mitochondrial membrane potential levels under the action of siPGC-1α;(10)By constructing a HCCLM3 cell nude mouse subcutaneous tumor-bearing model,and observing the related changes in tumor volume and size after treatment with CDDP and different calcium carbonate nanocarriers,the heart,liver,spleen,lung,and kidney were taken for HE staining,and the tumors were removed.The tissues were subjected to immunohistochemistry experiments to detect the expression of mitochondrial biogenesis,mitophagy,and apoptosis-related proteins.Results:(1)The effect of CDDP on the cell viability of Huh7 and HCCLM3 decreased in a concentration-dependent manner,and the IC50 were 3.514 μg/ml and 17.99 μg/ml;(2)The expression of mitochondrial autophagy-related proteins PINK1,Parkin and LC3-Ⅱ.was up-regulated with time-dependent,and the mitochondrial autophagy substrate p62 was down-regulated with time-dependently,which inhibited mitochondrial autophagy,decreased the cell viability of HCC cells,and significantly increased the level of apoptosis;(3)The relative mtDNA copy number increased in a time-dependent manner under CDDP,the expression of mitochondrial biogenesis-related proteins PGC-1α,NRF2,TFAM and mitochondria-associated protein TOM20 increased in a time-dependent manner,and the activity of HCC cells decreased and the level of apoptosis increased significantly after knockdown PGC-1α;(4)CDDP-ICG@CaCO3/PAA+siPGC-1α is spherical in shape with a particle size of 84.42±4.95 nm,which meets the basic conditions for EPR effect.The CDDP drug loading of CDDP-ICG@CaCO3/PAA+siPGC-1α was 6.68%,which showed an amorphous structure and had good photothermal conversion characteristics.It has good stability in water and different concentrations of BSA,and can be taken up by HCC cells.(5)The biological safety of CaCO3/PAA and ICG@CaCO3/PAA was high,and the killing effect of CDDP-ICG@CaCO3/PAA+siPGC-1α cells was strong,and the level of apoptosis was significantly increased,the expression of pro-apoptotic Bax protein increased in a time-dependent manner,and the anti-apoptotic protein Bcl-2,The expression of Mcl-1 decreased with time-dependence,and the expression of CleavedCaspase3 increased.(6)Under the action of CDDP-ICG@CaCO3/PAA+siPGC-1α,the expression of mitophagy-related proteins PINK1,Parkin,and LC3-Ⅱ was up-regulated at 24 hours,and then decreased at 48 hours.Mitophagy inhibitors the combined application of Mdivi-1 did not affect the cell viability of HCC cells;(7)Under the action of CDDP-ICG@CaCO3/PAA+siPGC-1α,the relative mtDNA copy number increased for a short period of time at 24 hours,but then gradually decreased.The expression of mitochondrial biogenesis-related proteins PGC-1α,NRF2,TFAM and mitochondrial-related protein TOM20 increased for a short period of time at 24 hours,and then gradually decreased;(8)Under the action of calcium carbonate nanocarriers,the relative mitochondrial Ca2+in HCC cells increased to varying degrees,among which the CDDP-ICG@CaCO3/PAA group was the most significant;(9)Under the action of calcium carbonate nanocarriers,the relative mtROS levels in HCC cells increased to varying degrees,among which the CDDP-ICG@CaCO3/PAA group was the most significant.CDDP and calcium carbonate nanocarriers can still reduce the mitochondrial membrane potential of HCC cells to a certain extent,CDDPICG@CaCO3/PAA is the most significan;(10)After CDDP-ICG@CaCO3/PAA+siPGC-1α treatment,the subcutaneous tumor volume of nude mice was significantly smaller than that of the Control group,and no structural damage was caused to important organs.The expression of mitochondrial biogenesis PGC-lα was weakened,the expression of mitophagy PINK1 was weakened,the expression of anti-apoptosis-related protein Bcl-2 was weakened,and the expression of Cleaved-Caspase3 was significantly enhanced.Conclusions:(1)Mitochondrial turnover pathways are involved in HCC resistance to cisplatin;(2)To address the chemotherapy resistance of HCC to cisplatin mediated by mitochondrial turnover pathways,we designed and prepared a calcium carbonate nanocarrier that interferes with the mitochondrial turnover pathway in HCC cells:CDDP-ICG@CaCO3/PAA+siPGC-1α;(3)We confirmed that CDDP-ICG@CaCO3/PAA+siPGC-1α can induce cell apoptosis by interfering with the mitochondrial turnover pathway and inducing mitochondrial Ca2+overload,thereby increasing the sensitivity of HCC to cisplatin.