Study Of The Inhibitory Effects Of β-hydroxyisovalerylshikonin On The Cervical Cancer Via PI3K/AKT/mTOR Signaling Pathway

Cervical cancer is a common malignant tumor of genital tract, while the pathogenesis is not yet completely understood. Clinically, the treatment options for cervical cancer are surgery, radiotherapy, chemotherapy and traditional Chinese medicine. Among them, the best one for treatment of cervical cancer is the multi-disciplinary comprehensive menthod combined with the advantages of various kinds of options mentioned above. Recent studies indicated that the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway regulates tumor cell proliferation, promotes cell cycle progression, participates in neovascularization, and induces tumor cells to resist radiotherapy and chemotherapy. Moreover, this signaling cascade is a major pathway in suppressing tumor cell apoptosis. Cervical cancer can be treated by triggering tumor cell apoptosis and combined application of radiotherapy and chemotherapy, which is a focus in molecular-targeted therapy of cervical cancer.Traditional Chinese medicine plays a unique role for the treatment of cervical cancer. Shikonin, a naphthoquinone derivative, is a naturally occurring active chemical found in the root of Lithospermum erythrorhizon, and has anti-cancer, anti-inflammation, anti-HIV virus and anti-fungus activities. Shikonin has strong toxicity, but does not exhibit tumor specificity and thus has not been widely used in the clinic. Studies of the extraction or synthesis of high-performance non-toxic shikonin derivatives has received great attention. P-hydroxyisovalerylshikonin (β-HIVS) is a natural shikonin derivative. Moreover, β-HIVS suppressed tumor cell proliferation, induced it apoptosis, and interfered with the cell cycle of tumor cells, highlighting its potential as a novel high-performance non-toxic antitumor medicine. Thus, on the basis of these in vitro and in vivo findings regarding the PI3K/AKT/mTOR signaling pathway, we acquire a deeper understanding of the occurrence, development, treatment and prognosis of cervical cancer, which would be a new strategy for molecular-targeted therapy of cervical cancer.1. Expressions of PTEN,Survivin,PI3K,AKT,mTOR,P70S6K in the progression of cervicalneoplasia and their clinical significances Objective To evaluate the expressions and clinical significances of PTEN, Survivin, PI3K, AKT, mTOR and P70 in the successive steps of progression in cervical lesions.Methods The expression of PTEN, Survivin, PI3K, AKT, mTOR, P70S6K were performed in a total of 146 patients with 10 normal cervical epithelium,20 cervical erosion metaplasia,60 cervical intraepithelial neoplasia (20 CIN Ⅰ,20 CIN Ⅱ,20 CIN Ⅲ), and 56 cervical squamous cell carcinomas were detected by SP immunohistochemistry, qRT-PCR and Western Blot.Results 1) PTEN expression progressively decreased along the continuum from cervical erosion metaplasia to cervical intraepithelial neoplasia and cervical squamous cell carcinoma(P<0.05); Survivin expression were progressively increased(P<0.05).PI3K/AKT/mTOR/P70S6K expression progressively increased along the continuum from normal epithelium to cervical erosion metaplasia,cervical intraepithelial neoplasia and cervical squamous cell carcinoma (P<0.05) 2) Positive PTEN immunostaining was associated with clinical stage(P<0.05), tumor size (P< 0.05) in cervical squamous cell carcinoma. Positive Survivin immunostaining was associated with clinical stage(P< 0.05), tumor size (P<0.05) and the differentiated degree (P<0.05). Positive PI3K/AKT/mTOR immunostaining was associated with clinical stage(P< 0.05), tumor size (P<0.05) in cervical squamous cell carcinoma. Positive P70 immunostaining was associated with clinical stage(P<0.05).3) There was a negative correlation between the expression of PTEN and Survivin in cervical cancer (r2=-0.613, P<0.01). P70S6K positive expression correlated significantly with PI3K,AKT and mTOR in cervical cancer (r10=0.418, P<0.01; r11=0.512, P<0.05; r12=0.365, P<0.01). PI3Kpositive expression correlated significantly with AKT and mTOR (r13=0.438, P<0.01; r14=0.366,P<0.01).AKT positive expression correlated significantly with mTOR(r15=0.351, P<0.01).4) qRT-PCR analysis showed that:compared to those in normal cervix uteri, PTEN mRNA in cervical carcinomas and CINⅢ were significantly decreased (P<0.01), Survivin mRNA in cervical carcinomas and CINⅢ were significantly increased (P<0.01), PI3K/AKT/mTOR/P70S6K mRNA in cervical carcinomas and CIN Ⅲ were significantly increased respectively (P<0.01), PI3K/AKT/mTOR/P70S6K mRNA in CIN Ⅱ were also increased respectively (P<0.05).5) Western Blot analysis showed that:compared to those in normal cervix uteri, PTEN in cervical carcinomas,CINⅢ,CIN Ⅱ and CIN Ⅰ were significantly decreased (P<0.01), Survivin in cervical carcinomas and CIN Ⅲ were significantly increased (P<0.01), Survivin in CIN Ⅱ were also increased (P<0.05), PI3K in cervical carcinomas,CIN Ⅲ,CIN Ⅱ and CIN Ⅰ were significantly increased (P<0.01), AKT/mTOR/P70S6K in cervical carcinomas and CINⅢ were significantly increased respectively (P<0.01), AKT/mTOR/P70S6K in CIN Ⅱ were also increased respectively (P<0.05). Conclusion PTEN,Survivin,PI3K,AKT,mTOR and P70S6K may be valuable biomarkers that are predictive of the progression and prognosis in patients with cervical squamous cell carcinoma. These useful biomarkers may have potential as promising therapeutic targets.2. β-hydroxyisovalerylshikonin induces human cervical cancer cell apoptosis via PI3K/AKT/mTOR signaling Objective To investigate the in vitro proliferation inhibitory effect of β-HIVS on human cervical cancer cells and to identify the mechanism of inducing HeLa cell apoptosis. Methods HeLa cells were treated with β-HIVS and the growth inhibitory effect was detected with MTT assay. Acridine orange/ethidium bromide staining was used for morphological changes in apoptotic HeLa cells. Annexin/propidium iodide staining was applied for HeLa cell apoptotic rate after treatment with P-HIVS. Flow cytometry was employed to analyze apoptosis and cell cycle distribution. qRT-PCR and western blot assays were used to examine PI3K/AKT/mTOR signaling pathway expression.Results 1) P-HIVS inhibited HeLa cell proliferation in a dose-and time-dependent manner.2) With increased concentration of β-HIVS, the apoptotic rate of HeLa cells was increased. Flow cytometry analysis showed that Hela cell cycle distribution changed when treated with β-HIVS. The counts of G0/G1 cells decreased, the cells in S phase increased, while the number of G2/ M phase cells had no obviously changed, which indicated that cell cycle was slightly arrested at S phase.3) Three different concentrations of β-HIVS evidently reduced PI3K, AKT, mTOR and P70S6K mRNA expression levels and PI3K, AKT, β-AKT, mTOR and P70S6K protein expression levels in HeLa cells after 48h in a time- and dose-dependent manner.Conclusion These results indicated that β-HIVS suppressed HeLa cell proliferation in a time-and dose-dependent manner. β-HIVS promoted cervical cancer cell apoptosis by inhibiting the PI3K/AKT/mTOR signaling pathway and suppressing downstream gene expression. Based on the signaling pathway of molecular targeted therapy is expected to develop a new way for cervical cancer treatment.3. In vivo anti-cervical cancer effects of P-hydroxyisovalerylshikonin through PI3K/AKT/mTOR signaling pathwayObjective To establish an animal model of cervical cancer on tumor-burdened nude mice in order to study the potential anti-tumor molecular mechanism of p-HIVS in vivo.Methods An animal model of cervical cancer on tumor-burdened nude mic was established. And then, the macroscopic measurements were taken to evaluate inhibitory effects of p-HIVS treated for the subcutaneously transplanted tumors in nude mice. We detected morphological changes via HE staining, apoptosis status with Tunel staining method. qRT-PCR and Western blot were used to detect the mRNA and protein expressions of PI3K, AKT, mTOR and P70S6K after P-HIVS treatment.Results 1) The growth rate of cervical cancer transplantation tumor that treated by P-HIVS was obviously lower than the control group. Both weight and volume of the tumors in drug injected group were significantly smaller than the control one(p< 0.05). P-HIVS could inhibit the proliferation of human cervical cancer (tumor inhibition rate was 32.80%).2) Histological HE staining showed that the tumor cells of p-HIVS treatment group became degeneration and necrosis, which the nest structure loosened. Cell nuclears turned to pyknosis or karyorrhexis, and the condensed chromatins dispersed around the nucleus or formed the crescentric structures.3) The typical apoptotic morphological changes were found by the Tunel staining method.4) qRT-PCR demonstrated that the mRNA expressions of PI3K, AKT, mTOR and P70S6K were decreased after treated by P-HIVS than the control respectively (p<0.01). Western blot demonstrated that the protein expressions of PI3K, AKT, p-AKT, mTOR and P70S6K were decreased than the control respectively (p<0.05).Conclusion Through inducing tumor cell apoptosis, P-HIVS could obviously inhibit the growth of cervical cancerous tissue in vivo. The PI3K/AKT/mTOR signaling pathway may play an important role in inducing the cervical cancer cells apoptosis that treated by P-HIVS.