key: cord-0075161-kalla3di authors: Zhang, Fan; Zhang, Hua; Qian, Wenjun; Xi, Yuyan; Chang, Lihua; Wu, Xiaoling; Li, Mu title: Matrine exerts antitumor activity in cervical cancer by protective autophagy via the Akt/mTOR pathway in vitro and in vivo date: 2022-02-07 journal: Oncol Lett DOI: 10.3892/ol.2022.13230 sha: 49ec5fdce54ca5a001dfa6acb4d48e55af3a0ec7 doc_id: 75161 cord_uid: kalla3di Matrine is a quinazoline alkaloid extracted from Sophora flavescens. The aim of the present study was to determine whether matrine can induce autophagy in the human HeLa and SiHa cervical cancer cell lines in vitro and in vivo. Cell viability assay was used to assess the suppressive effect of matrine and cisplatin on the proliferation of HeLa and SiHa cells. A total of 28 4-week-old female BALB/c nude mice were used for the in vivo study. Autophagy and protein expression were observed via transmission electron microscopy, monodansylcadaverine and immunohistochemical staining and western blotting. The inhibitory effect of matrine on the proliferation of cervical cancer cells was time- and dose-dependent. The combination of matrine and cisplatin synergistically inhibited the proliferation of cervical cancer cells in vitro and in vivo. Transmission electron microscopy showed that after the addition of matrine, numerous autophagosomes and autophagolysosomes were observable in HeLa and SiHa cells, as demonstrated by monodansylcadaverine staining. Western blotting and immunohistochemical staining showed that as the concentration of matrine increased, the expression of the autophagy marker LC3A/B-II also increased significantly in vitro and in vivo. These findings suggested that matrine inhibited the proliferation of cervical cancer cells and induced autophagy by inhibiting the Akt/mTOR signaling pathway. Thus, matrine may represented a potential candidate in combination therapy for cervical cancer as an inducer of autophagy. Cervical cancer was the fourth most frequently diagnosed cancer worldwide in 2018, with an estimated 570,000 new cases. It was also the fourth leading cause of cancer-related deaths in women worldwide, accounting for an estimated 311,000 deaths in 2018 (1) . In China, the large size of the population constitutes a great challenge to cervical cancer screening and vaccination programs (2) . Despite a current standard of care that includes radiotherapy, chemotherapy and/or surgical resection (3), the 5-year survival of patients with advanced recurrent and metastatic cervical cancer is only 15% (4) . To achieve satisfactory therapeutic effectiveness for patients with advanced cervical cancer, improved treatment strategies are needed. Matrine is a quinazoline alkaloid extracted from the dried roots and fruits of Sophora flavescens (5) , and preparations for injection and for use as alkaloid gels have been clinically used in China for several years to treat gynecological inflammation, gynecological tumors, hepatitis and other diseases (6) (7) (8) . The main component of Sophora flavescens preparations is matrine, which has broad pharmacological properties, including sedation, analgesia (9) , antiviral (10), anti-inflammatory (11) , antifibrotic (12) antitumor and immunoregulatory activity (13, 14) , as well as other effects (15) . Previous studies have investigated the inhibitory effects of matrine on a variety of tumor cells including cervical cancer (16) (17) (18) (19) (20) . Matrine combined with chemotherapy drugs, such as cisplatin, docetaxel and adriamycin, may have antitumor activity (21) (22) (23) . Matrine has also been shown to enhance the levels of immunological parameters such as IgA, IgM, CD8 + and IFN-γ, improve cachexia in patients with advanced cancer, and reduce adverse reactions caused by platinum-based chemotherapy (24) . Autophagy is ubiquitous in tumor cells, and during various stages of the development and progression of cervical cancer, autophagy promotes cell death or cell survival (25) . Matrine also has a complex, multifaceted role in tumor development, and can either promote or inhibit autophagy. Matrine-induced autophagy may have opposite effects in different types of tumor cells (26, 27) . Previous studies have reported that matrine can induce autophagy, promote cell death and enhance the sensitivity of cancer cells to some chemotherapeutic drugs (28, 29) . Others have reported opposite effects, including reduced autophagy and increased cancer cell survival (30) . Our previous observations suggested that matrine could inhibit the growth of cervical cancer by downregulating the p38 signaling pathway (19) . However, whether matrine can induce autophagy in cervical cancer remains unclear. In the present study, matrine inhibited the proliferation of cervical cancer cells in vitro and suppressed tumor growth in vivo. With the increased matrine concentration, the expression of the autophagy marker LC3A/B-II increased both in vitro and in vivo, and this effect was mediated by the Akt/mTOR pathway. These findings suggest that matrine-induced autophagy may prove useful for the treatment of cervical cancer. Reagents. Tumor xenograft model. The Institutional Animal Care and Treatment Committee of Xi'an Jiaotong University approved the study (approval no. XJTULAC2020-1337). A total of 28 4-week-old female BALB/c nude mice (weight 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were kept in a specific-pathogen-free laminar air flowbox at 22˚C, humidity at 30-50% and light/dark cycle of 14/10 h and were fed with sterile food pellets and water ad libitum. After HeLa cells in logarithmic growth phase were digested with trypsin, a cell suspension was prepared. HeLa cells (1x10 6 ) suspended in 0.2 ml PBS were implanted subcutaneously into the right flank of the nude mice. After 7 days, the mice were randomly assigned to the following groups (n=7/group): i) Control, administered 100 µl normal saline; ii) matrine 50 mg/kg; iii) cisplatin 2 mg/kg; and iv) matrine 50 mg/kg + 2 mg/kg cisplatin. The mice were treated by intraperitoneal injection once every 2 days and were sacrificed 21 days later. The length (l) and width (w) of the tumor were measured every 2 days and the volume (V=l x w 2 /2) was calculated. The mice were sacrificed by cervical dislocation after 21 days, and the tumors were excised and weighed. The proliferation of HeLa and SiHa cells was assessed using CCK-8 assays. As shown in Fig. 1B , matrine inhibited the proliferation of HeLa and SiHa cells. Indeed, matrine inhibited the proliferation of HeLa and SiHa cells in both a dose-(0-10 mM) and time-(24 and 48 h) manner. Numerous cytoplasmic vacuoles of varying size were present in HeLa and SiHa cells treated with matrine after 24 h. In addition, the number of small, rounded cells increased when the matrine concentration was increased (Fig. 1C) . Transmission electron microscopy revealed an abundance of autophagosomes and autophagolysosomes in HeLa and SiHa cells treated with 5 mM matrine ( Fig. 2A) compared with untreated control cells. Consistent with the evidence from transmission electron micrographs, MDC staining revealed an abundance of positive particles in HeLa and SiHa cells treated with matrine compared with untreated control cells (Fig. 2B) . Western blot assays found that the expression of LC3A/B-II increased significantly as the dose of matrine and the duration of treatment increased (Fig. 2C) . These results suggested that matrine induced autophagy in cervical cancer cells in vitro. Matrine initiates autophagy by activating the Akt/mTOR signaling pathway. The effect of matrine treatment on Akt/mTOR signaling pathway was assessed by analyzing the expression of Akt, p-Akt, mTOR, p-mTOR and downstream proteins via western blotting. Matrine inhibited the phosphorylation of mTOR-Ser 2448, p70S6K (a downstream target of mTOR) Akt-Ser 473 (an upstream regulator of mTOR) (Fig. 3) . These findings suggested that matrine promoted HeLa and SiHa cell autophagy via the Akt/mTOR signaling pathway. Matrine and cisplatin synergistically inhibit cervical cancer cell proliferation in vitro. Because matrine alone inhibited the proliferation of cervical cancer cells, the next experiments aimed to determine its effects when combined with cisplatin were investigated. Following treatment for 24 h, the IC 50 of matrine was 2.181 mM for HeLa cells and 2.178 mM for SiHa cells. The corresponding IC 50 values for cisplatin were 0.008 and 0.009 mM ( Fig. 4A and C) . When matrine and cisplatin were combined (1:300) for 24 h treatment, the inhibition of HeLa and SiHa cell proliferation was greater than that seen with either drug alone. The corresponding IC 50 values for combination group were 0.612+0.002 mM for HeLa cells and 0.587+0.002 mM. An affected fraction (Fa) of 0.5 corresponds to 50% survivability. At Fa=0.5, the CI was 0.26 for HeLa and 0.34 for SiHa cells (Fig. 4B and D) . These values suggested that the combination of matrine and cisplatin had a synergistic effect in HeLa and SiHa cells. Tumor growth following HeLa cell transplantation into nude mice and treatment with matrine. BALB/c nude mice were used to establish an cervical tumor xenograft model and to determine whether matrine induced autophagy in vivo. Matrine (50 mg/kg) + cisplatin (2 mg/kg) significantly suppressed tumor growth (Fig. 5A ) without leading to a reduction in body weight (Fig. 5B) . The volume (Fig. 5C ) and weight (Fig. 5D) (Fig. 5E ). In addition, immunohistochemical staining ( Fig. 5E and F) and western blotting ( Fig. 5G and H) suggested that both matrine and matrine + cisplatin upregulated LC3A/B-II in tumor tissue. The global prevalence of cervical cancer has dropped from the third place 10 years ago to the fourth place in 2018 (1). The change is associated with the development of medical standards in some countries, increased cervical cancer screening and human papillomavirus vaccination coverage and enhancement of women's health awareness. These interventions seem oncologically safe in the current selection of patients with low-stage and low-grade disease (32) . Internationally, the chance for recurrence in early stage cervical cancer is 6-9% (33). However, patients with special types of cervical cancer, such as mid-to-late stage, relapsed, drug-resistant disease, and fertility needs, require multidimensional, multiplan, individualized treatment with few side effects and palliative treatment when necessary (34) . Improved understanding of the role and mechanism of matrine in HeLa and SiHa cells may provide new ideas for the treatment of cervical cancer (35) . Cisplatin is the first-line drug in adjuvant combined chemotherapy regimens for cervical cancer (36) . Drugs that act synergistically with and reduce the toxicity of cisplatin are important (37) . The inhibitory effects of matrine and cisplatin were stronger than those either drug alone (38) . When Fa=0.5, isobologram analysis determined that the CI of the drugs in combination was <1.0, indicating that the two drugs had a synergistic effect. The effects of matrine and cisplatin have also been found to be synergistic in urothelial bladder cancer and rhabdomyosarcoma cells (21, 39) . Matrine may therefore increase the sensitivity of cervical cancer cells to cisplatin. In the present study, matrine combined with cisplatin significantly inhibited tumor volume and weight in vivo. The in vivo antitumor activity of matrine combined with cisplatin has been reported in liver cancer (16) , and the combination may be less toxic to normal cells (40) . Sophora flavescens has been widely used clinically in China to treat various diseases including tumors (41) . Moreover, matrine suppositories or Sophora flavescens alkaloid gels are used for gynecological diseases such as trichomonal vaginitis, candidal vaginitis and chronic cervicitis (6) . Matrine injection is also used for adjuvant treatment of chronic hepatitis (42) . Surprisingly, Matrine and Sodium Chloride Injection have a good clinical effect in the treatment of the model mice and 40 patients with coronavirus disease 2019 (COVID-19) (43) . At present, the most commonly used anti-tumor matrine drug in clinical practice is Compound Kushen Injection in traditional Chinese medicine (31, 44, 45) . It is a mixture of natural compounds extracted from two medical herbs: Sophora flavescens and Rhizoma smilacis Glabrae (7). Our previous study have suggested that matrine inhibits the proliferation of HeLa and C33A cells (19) . Additionally, matrine combined with cisplatin shows antitumor activity in U14 rats with cervical cancer (46) . Thus, matrine has the potential for development as therapeutic or adjuvant agent for human cervical cancer. However, matrine as single dosage in the treatment of cervical cancer is still unconfirmed. The reason may be that their ability to inhibit tumor proliferation is limited. In addition, the specific mechanism underlying their potential anti-tumor effects remain unclear. The findings of the present study provide an updated perspective on the relationship between the antitumor property of matrine (either alone or combined with cisplatin) and autophagy, as well as its underlying molecular mechanism. Autophagy is a double-edged sword in cancer treatment (47) (48) (49) . Recent studies have shown that the anticancer activity of matrine involves promotion of autophagy (29, (50) (51) (52) . In the present study, both transmission electron microscopy and MDC staining revealed the formation of numerous autophagosomes and autophagolysosomes in cervical cancer cells treated with matrine. In cervical cancer cells, matrine promoted autophagy, as suggested by upregulation of LC3A/B-II in vitro and in vivo. However, the increase in LC3A/B-II cannot be entirely attributed to the increase of autophagy and formation of autophagosomes, but also to decreased digestion of substances in autophagolysosomes. Therefore, it is necessary to determine whether autophagy was inhibited and whether the increase in LC3A/B-II was the result of an increase in autophagy. The Akt/mTOR signaling pathway is known to be associated with autophagy (53-55). Akt-mediated mTORC1 activation is a key regulator of cell anabolism and growth, and p70S6K is a downstream target of mTORC1 (56) . The Akt/mTOR signaling pathway is also active in human papilloma virus (HPV) infection (57) . HPV activates the mTORC1 complex (58), increases protein synthesis and reduces host cell autophagy before HPV-16 enters keratinocytes in the early stage to protect the virus entering the body from rapid degradation, thereby prolonging the lifespan of HPV in the host cell (59) . In several malignancies, the anticancer activity of matrine depends on inducing autophagy by blocking the Akt/mTOR signal cascade (52, (60) (61) (62) . In the present study, matrine inhibited the phosphorylation of Akt (Ser 473), mTOR (Ser 2448) and the downstream effector molecule p70S6K (Thr 389). Overall, these results are consistent with previous studies on other tumor types (52, 61, 62) . In conclusion, matrine inhibited the growth of cervical cancer cells by enhancing autophagy in vitro and in vivo. The synergy between matrine and cisplatin provide a theoretical basis for the clinical application of combined treatment of cervical cancer. In addition, matrine-induced autophagy may involve the Akt/mTOR signaling pathway. 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The data sets generated and/or analyzed during the present study are available from the corresponding author on reasonable request. FZ, ML and XW conceived and designed the study. FZ, HZ, WQ, LC and YX performed the in vitro and in vivo experiments and analyzed the data. FZ wrote the manuscript. ML, XW, and LC helped modify the manuscript. FZ revised the manuscript. FZ and HZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript. Experiments involving animals were approved by The Biomedical Ethics Committee of The Medical Department of Xi'an Jiaotong University (approval no. XJTULAC2020-1337). Not applicable. The authors declare that they have no competing interests.