Hirsutine Induces Apoptosis In Human Lung Cancer Cells Through ROCK1/PIEN/PI3K/GSK3? Pathway

BackgroundLung cancer is one of the largest cancer in China,and it is of great significance for us to search for novel high-efficiency,low-toxicity anti-lung cancer drugs.Natural alkaloids are important source of antitumor drugs,and we screened a large number of anti-tumor activities of natural alkaloids in our preliminary experiments.As a result,we found that Hirsutine possesses high anti-lung cancer activity in vitro and in vivo.However,it exerts low toxicity for normal cells,suggesting that Hirsutine has the potential to be developed as a novel drug against lung cancer with high efficiency and low toxicity.Studies have reported that Hirsutine is one of the alkaloid active ingredients in Uncaria rhynchophylla.Otherwise,it was also reported to possess the ability of protecting hypoxia-induced myocardial injury and protecting against inflammation-mediated neuronal damage.Meanwhile,Hirsutine also can inhibit the metastasis and invasion and induce DNA damage in breast cancer cells,suggesting that Hirsutine has not only anti-tumor activities but also protective effects to human's body.However,the anti-lung cancer effect and molecular mechanisms of Hirsutine remains unclear.Therefore,the present study aims to elucidate whether Hirsutine has selective anti-lung cancer effects and further clarify the underlying molecular mechanisms.1.Hirsutine selectively induces apoptosis in lung cancer cellsCCK8 and flow cytometry showed that Hirsutine induced apoptosis in lung cancer A549 cells and NCI-H1299 cells in dose-and time-dependent manner.Western blot analysis revealed that Hirsutine increased the expression of apoptosis-related proteins Cleaved PARP?C-PARP?and Cleaved-Caspase-3?C-CASP 3?.In contrast,Hirsutine exerted little toxicity toward human normal liver cell LO2 and human normal embryonic lung cell WI-38.All these findings indicate that Hirsutine selectively induces apoptosis in human lung cancer cells but not normal lung fibroblast cells and normal hepatocytes.2.Hirsutine induces mitochondrial injury in lung cancer cellsTransmission electron microscopy showed that the mitochondria were vacuolization,swelling and internal hemorrhoids under Hirsutine treatment,suggesting that Hirsutine induced mitochondrial damage in A549 cells.JC-1 staining showed that Hirsutine significantly down-regulated mitochondrial membrane potential in a dose-dependent manner.The luciferase ATP assay showed that Hirsutine significantly down-regulated the levels of cellular ATP.DCFH-DA staining and flow cytometry assay revealed that Hirsutine significantly increased ROS production.Western blot assay also confirmed that Cyto C was released from mitochondria into the cytosol in response to Hirsutine treatment.Calcein-AM/CoCl₂ staining revealed that Hirsutine induced the opening of mitochondrial permeability transition pore?MPTP?,immunoprecipitation assasy and the employment of cyclosporin A?CSA?,an MPTP open inhibitor,confirmed that Hirsutine promoted cyclophilin D?CypD?and adenylate transporter?adenine nucleotide translocase 1,ANT1?binds directly,finally promoting the opening of MPTP,leading to mitochondrial-dependent apoptosis.3.Hirsutine induces apoptosis of lung cancer cells via ROCK1/PTEN/PI3K/GSK3?pathwayIn order to further explore the molecular mechanisms underlying Hirsutine-induced mitochondrial injury in lung cancer cells,Western blot assay was performed and the results showed that Hirsutine induced GSK3??S9?dephosphorylation in dose-and time-dependnet manner.Pretreatment cells with CHIR99021,a GSK3?inhibitor,further confirmed that Hirsutine-induced MPTP opening is dependent on GSK3??S9?dephosphorylation-mediated ANT1/CypD interaction.Since GSK3?is an important downstream target of PI3K/Akt pathway,we further determined the effects of hirsutine on phosphorylation of PI3K and Akt.Treatment of A549cells with hirsutine resulted in decreases in levels of phospho-PI3K?p-PI3K?and phospho-Akt?p-Akt?in a dose-and time-dependent manner.Pretreatment with LY294002?PI3K inhibitor?markedly enhanced hirsutine-mediated Akt inactivation and GSK3?dephosphorylation.Pretreatment with LY294002 significantly enhanced hirsutine-mediated inhibition of phospho-GSK3?/ANT1 interaction,resulting in an increase in the association between CypD and ANT1,finally causing mPTP opening and apoptosis.In addition,we found that Hirsutine induced ROCK1 activation and PTEN phosphorylation.Both ROCK inhibitor Y-27632 and lentiviral gene knockdown of ROCK1blocked Hirsutine-induced PTEN phosphorylation,PI3K/Akt/GSK3?dephosphorylation.Furthermore,pretreatment cells with Y-27632 or knockdown of ROCK1 also attenuated hirsutine-mediated inhibition of phospho-GSK3?/ANT1 interaction,ANT1/CypD interaction,mPTP opening,ATP depletion,and apoptosis.All of these findings suggest that ROCK1/PTEN/PI3K/Akt signaling pathway plays a crucial role in hirsutine-mediated GSK3?dephosphorylation,ATP depletion and mitochondrial apoptosis.4.Hirsutine inhibits tumor growth and induces apoptosis in an A549 xenograft mouse model through ROCK1/PTEN/PI3K/Akt/GSK3?signaling pathwayNude mice were inoculated subcutaneously with A549 cells and injected with either vehicle or Hirsutine,Tumor volume and body weight were measured every week,and the behavior of mice was observed daily.Treatment of mice with hirsutine resulted in a significant suppression of tumor growth compared with the control group.However,there were no significant differences in body weight between the two groups of mice.H&E staining of liver and kidney revealed no morphological difference between the control and Hirsutine-treated animals,suggesting that Hirsutine selectively inhibits tumor growth in A549 xenograft mouse model without causing additional toxic effect on normal tissues in a certain dose and time.To verify the important role of the ROCK1/PTEN/PI3K/Akt/GSK3?pathway in Hirsutine-induced anticancer effect in vivo,we detected the expression of ROCK1,p-PTEN,p-PI3K,p-Akt and p-GSK3?using immunohistochemistry and Western blot assay.The immunohistochemical staining showed that the expression of C-Caspase 3 upregulated and the level of p-GSK3?decreased in Hirsutine treatment group.Western blot analysis conducted on excised tumor tissue revealed that treatment with hirsutine resulted in cleavage/activation of ROCK1,activation of PTEN,inactivation of PI3K,Akt and dephosphorylation of GSK3?.Together,these results indicate that hirsutine effectively inhibits tumor growth in an A549 xenograft mouse model through ROCK1/PTEN/PI3K/Akt/GSK3?pathway.In summary,the present study indicates that Hirsutine effectively induces apoptosis of a variety of lung cancer cells and inhibits tumor growth in a lung cancer xenograft mouse model.Mechanism studies revealed that the anticancer effect of Hirsutine is dependent on the activation of the ROCK1/PTEN/PI3K/Akt/GSK3?pathway,which in turn causes MPTP open-mediated mitochondrial injury and ultimately leads to caspase activation and apoptosis.This study has clarified the anticancer effect of Hirsutine in vitro and in vivo,and elucidated the molecular mechanisms of Hirsutine against lung cancer,and provided experimental basis for Hirsutine being developed as a highly effective,low-toxic anti-lung cancer drug and promoting ROCK1/PTEN/PI3K/Akt/GSK3?pathway to be developed as a novel target for cancer treatment.