The Mechanism Of 3,3 ’-diindolylmethane Inhibiting Proliferation Of Gastric Cancer Cells Through STIM1-mediated SOCE

Objective3,3’-diindolylmethane（DIM）is a natural phytochemicals obtained in cruciferous Brassica vegetables such as broccoli,cabbage,cauliflower and kale.Previous research in our laboratory have found that DIM have significantly inhibited growth in gastric cancer cells,but its specific mechanism is still unclear.Therefore,in this study,we took human BGC-823 and SGC-7901 gastric cancer cell lines as the research objects to explore the underlying mechanism of anti-cancer effect of DIM in-depth,which may provide experimental evidence for the development and clinical application of natural phytochemicals DIM for gastric cancer prevention and treatment in the future.Methods1.Treating the human BGC-823 and SGC-7901 gastric cancer cell lines with different concentration of DIM（0,10,20,40,60,80,100,120 μM）for 24 h or 48 h,then MTT assay was used to detect cells viability.2.With treating different concentration of DIM（0,20,40,60,80 μM）for 24 h in human BGC-823 and SGC-7901 gastric cancer cells,hoechst 33342 staining and flow cytometry analysis were to measure cells apoptosis level,western blot was to detect apoptosis-related proteins Bcl-2 and Bcl-2 associated X protein（Bax）,cleaved-caspase 3 and autophagy-related protein LC3II/LC3 I.3.To explore the molecular mechanism in anti-cancer effect of DIM,DIM was used to treat BGC-823 gastric cancer cells,high-throughput sequencing was to detect expression of CAAT/enhancer-binding protein（CHOP）m RNA compared to control group,the proteins expression of ER stress-related proteins phospho-Inositol requiring enzyme 1（p-IRE1α）,phospho-Protein kinase-like ER kinase（p-PERK）,Activating transcription factor 6（ATF6）,CHOP was detected by western blot.After BGC-823 gastric cancer cells were respectively transfected with CHOP siRNA or ATF6 siRNA for 48 h and DIM was treated for another 24 h,MTT assay was to detect cells viability and western blot to detect expression level of protein to explore the role of ER stress in anti-cancer effect of DIM.4.To further explore regulatory mechanism of the upstream AMPK signaling pathway,western blot method was used to detect the expression level of Adenosine 5’-monophosphate（AMP）-activated protein kinase（AMPK）signaling pathway related proteins phospho-AMP-activated protein kinase（p-AMPK）and phospho-Acetyl Co A carboxylase（p-ACC）after treating with different concentration of DIM（0,20,40,60,80 μM）.Pretreating cells with p-AMPK inhibitor Compound C for 1 h and then co-treating with DIM for another 24 h,the cells viability was tested by MTT assay and the expression of proteins by western blot,which to explore the role of AMPK signaling pathway in anti-cancer effect by DIM.5.In order to clarify the role of Ca²⁺ in the process of DIM inhibiting development in gastric cancer,after treating with different concentration of DIM（0,20,40,60,80 μM）for 24 h in human BGC-823 and SGC-7901 gastric cancer cell lines,Ca²⁺ fluorescence intensity was monitored under a laser scanning confocal microscope and expression of Calpain protein by western blot.Cells were pretreated with pharmacological inhibitor BAPTA-AM for 0.5 h and then co-treated with DIM for 24 h,MTT was used to examine cells viability and western blot to expression of related proteins.6.To further determine regulatory mechanism in DIM-induced cytoplasmic Ca²⁺ overload,laser scanning confocal microscope monitored ER calcium store Ca²⁺ fluorescence intensity and western blot was tested stromal interaction molecular 1（STIM1）expression after treating cells with different concentration of DIM（0,20,40,60,80 μM）for 24 h.STIM1 siRNA was used to interfere with cells for 48 h or the pharmacological inhibitor 2-APB to pretreated cells for 0.5 h and then co-treated with DIM for 24 h,MTT method was to measure cells viability and western blot to protein expression,which explore the role of STIM1-mediated Store-operated Ca²⁺ entry（SOCE）in anti-cancer effect by DIM.Results1.DIM significantly inhibited cells proliferation of human BGC-823 and SGC-7901 gastric cancer cell lines in a concentration and time dependent manner.2.Hoechst 33342 staining revealed that the cells nucleus was pyknotic,fragmented and the fluorescence intensity increased after DIM treatment.The flow cytometry results showed that DIM significantly increased the apoptotic cells population.Western blot showed that DIM significantly increased the expression of apoptosis-related proteins cleaved-caspase 3,Bax and autophagy-related protein LC3 II as well as decreased anti-apoptotic protein Bcl-2 in a concentration-dependent manner.3.High-throughput sequencing and western blot showed that DIM could significantly up-regulate the expression of CHOP,suggesting that ER stress is activated by DIM.Moreover,DIM significantly increased ATF6,but not p-IRE1α and p-PERK.After cells were transfected with CHOP siRNA or ATF6 siRNA,ER stress was inhibited,DIM-induced proliferation inhibition and apoptosis as well as autophagy was attenuated.4.DIM significantly increased expression of p-AMPK/p-ACC,indicating AMPK signaling pathway was activated.The combination of Compound C and DIM could reverse effect of proliferation inhibition and apoptosis as well as autophagy,moreover,ER stress also suppressed.5.DIM could significantly increase cytoplasmic free Ca²⁺ level and Calpain expression in a concentration dependent manner.After combination BAPTA-AM with DIM,p-AMPK-mediated ER stress activation by DIM was inhibited,the proliferation inhibition and apoptosis as well as autophagy were also restored.6.DIM could weaken ER calcium store Ca²⁺ fluorescence intensity and up-regulate STIM1 expression in a concentration dependent manner,indirectly suggesting that DIM could activate STIM1-mediated SOCE to induce cytoplasmic free Ca²⁺ overload.In addition,with the knockdown of STIM1 or non-specific inhibition of SOCE reducing Ca²⁺ influx and cytoplasmic Ca²⁺,DIM-induced p-AMPK/p-ACC mediated ER stress,proliferation inhibition,apoptosis and autophagy were suppressed.ConclusionDIM could significantly reduce human BGC-823 and SGC-7901 gastric cancer cell lines proliferation level and induce apoptosis as well as autophagy in a concentration-dependent manner;its mechanism may be related to STIM1-mediated SOCE activation and the downstream p-AMPK-mediated ER stress,DIM could decrease the level of ER calcium store Ca²⁺ and increase the expression of SOCE essential protein STIM1,indirectly suggesting that DIM could significantly activate SOCE,which subsequently induced ER Ca²⁺ release,extracellular Ca²⁺influx,and thus increase cytoplasmic Ca²⁺ level.After intervening in the activation of STIM1-mediated SOCE from the genetic and pharmacological level,the effect of proliferation inhibition and apoptosis as well as autophagy induced by DIM were restored.Furthermore,the activation of downstream AMPK signaling pathway and ER stress by DIM were also inhibited,indicating that DIM inhibited gastric cancer cells proliferation and induced apoptosis as well as autophagy through mediating STIM1-mediated SOCE induced the activation of downstream p-AMPK/ER stress.In summary,STIM1-mediated SOCE is a key regulatory target of DIM to inhibit the growth of gastric cancer,and effective enhancement of STIM1-mediated SOCE could significantly enhance the anti-gastric cancer effect of DIM,in addition,it should be attentive to recommend the patients with cancer taking anti-cancer drugs acting on STIM1-mediated SOCE with synergistic effect.