Studies Of The Pharmacological Effects Of Baicalin,an Extract From Scutellaria Baicalensis,on Bladder Cancer

Background:Ferroptosis is a new non-apoptotic modality of regulatory cell death,which is driven by iron-dependent,abnormal increase of lipid oxygen free radicals and imbalance of redox homeostasis.Previous studies have demonstrated that ferroptosis has been implicated in the onset and development of numerous diseases,including ischemia-reperfusion injury,cardiovascular diseases,acute kidney injury and neurodegenerative disorders.Recently,increasing studies have confirmed that ferroptosis contributes to eliminate cancer cells in an apoptosis-independent manner.So,ferroptosis is attracting more and more attention due to its effectiveness in cancer treatment.Scutellaria baicalensis Georgi or Huangqin,as a medicinal plant,is used widely in many Asian countries especially in China.Baicalin,a monomer of Chinese traditional herbs extracted from Scutellaria baicalensis Georgi,has been discovered its antitumor effects on several tumor progression.However,its function in bladder cancer and relationship with ferroptosis remain unexplained.Methods:We conducted a series of tests like cell viability assay,colony formation assay,apoptosis assays,mitochondria mass assessment,q PCR,western blotting,determination of ROS level and Fe²⁺level after different treatments to detect the function of baicalin.Results:In this study,we found that baicalin can induce cell death in bladder cancer cells in a dose-dependent manner.and this result was verified by apoptosis assay and colony formation.In order to explore the main signaling pathway of baicalin,we combined baicalin with different cell death pathway inhibitors（ferroptosis inhibitor DFO,ROS inhibitor NAC,glutathione supplement GSH,GPX4inhibitor RSL3,necrosis inhibitor Nec-1,autophagy inhibitor CQ and pan-caspase inhibitor Z-VAD-FMK）to treat bladder cancer cells.We found that autophagy,necrosis and pancaspase inhibitors did not decrease the efficacy of baicalin,while ferroptosis inhibitor DFO could significantly reverse the efficacy of baicalin,indicating that ferroptosis was the main cause of baicalin induced cell death.At the same time,we found that supplementation of glutathione GSH and inhibiting ROS by NAC can also reduce baicalin induced cell death.In order to explore the way of depletion of GSH,we combined the iron death agonist RSL3 and erastin,found RSL3increased the efficacy of baicalin,illustrating the baicalin promoted ROS,increased GSH consumption thus reduce intracellular GSH levels.Through Fe²⁺concentration assay,ROS level deter mination,WB and other methods,we verified that DFO and RSL3 could influence the efficacy of baicalin respectively.In terms of molecular mechanism,we demonstrated that FTH1 was an important factor in baicalin induced ferroptosis.Overexpression of FTH1 inactivated the anticancer effect of baicalin in bladder cells.Knocking down FTH1 boosted baicalin’s potence.Subcutaneous tumor-formation experiments in nude mice showed that baicalin could inhibit tumor growth,while combined with DFO showed the antitumor effect of baicalin.And under the experiment concentration of DFO and baicalin had no toxicity on body weight of nude mice.Tumor tissue of Prussian blue staining and immunohistochemical staining results showed that baicalin can obviously increase the Fe³⁺levels and inhibit the expression of ferritin,which process could be iron die inhibitors DFO block.Conclusions:Baicalin,the active component of Astragalus membranaceus,can inhibit the proliferation of bladder cancer by inducing FTH1-dependent ferroptosis,and is expected to be a potential compound for the treatment of bladder cancer.