Tumor immunity: A brief overview of tumor­infiltrating immune cells and research advances into tumor­infiltrating lymphocytes in gynecological malignancies (Review).

Tumor immunity is a promising topic in the area of cancer therapy. The 'soil' function of the tumor microenvironment (TME) for tumor growth has attracted wide attention from scientists. Tumor-infiltrating immune cells in the TME, especially the tumor-infiltrating lymphocytes (TILs), serve a key role in cancer. Firstly, relevant literature was searched in the PubMed and Web of Science databases with the following key words: 'Tumor microenvironment'; 'TME'; 'tumor-infiltrating immunity cells'; 'gynecologic malignancies'; 'the adoptive cell therapy (ACT) of TILs'; and 'TIL-ACT' (https://pubmed.ncbi.nlm.nih.gov/). According to the title and abstract of the articles, relevant items were screened out in the preliminary screening. The most relevant selected items were of two types: All kinds of tumor-infiltrating immune cells; and advanced research on TILs in gynecological malignancies. The results showed that the subsets of TILs were various and complex, while each subpopulation influenced each other and their effects on tumor prognosis were diverse. Moreover, the related research and clinical trials on TILs were mostly concentrated in melanoma and breast cancer, but relatively few focused on gynecological tumors. In conclusion, the present review summarized the biological classification of TILs and the mechanisms of their involvement in the regulation of the immune microenvironment, and subsequently analyzed the development of tumor immunotherapy for TILs. Collectively, the present review provides ideas for the current treatment dilemma of gynecological tumor immune checkpoints, such as adverse reactions, safety, personal specificity and efficacy.

Paeonol repurposing for cancer therapy: From mechanism to clinical translation.

Paeonol (PAE) is a natural phenolic monomer isolated from the root bark of Paeonia suffruticosa that has been widely used in the clinical treatment of some inflammatory-related diseases and cardiovascular diseases. Much preclinical evidence has demonstrated that PAE not only exhibits a broad spectrum of anticancer effects by inhibiting cell proliferation, invasion and migration and inducing cell apoptosis and cycle arrest through multiple molecular pathways, but also shows excellent performance in improving cancer drug sensitivity, reversing chemoresistance and reducing the toxic side effects of anticancer drugs. However, studies indicate that PAE has the characteristics of poor stability, low bioavailability and short half-life, which makes the effective dose of PAE in many cancers usually high and greatly limits its clinical translation. Fortunately, nanomaterials and derivatives are being developed to ameliorate PAE's shortcomings. This review aims to systematically cover the anticancer advances of PAE in pharmacology, pharmacokinetics, nano delivery systems and derivatives, to provide researchers with the latest and comprehensive information, and to point out the limitations of current studies and areas that need to be strengthened in future studies. We believe this work will be beneficial for further exploration and repurposing of this natural compound as a new clinical anticancer drug.

Effect of integrin ß1 in the treatment of stress urinary incontinence by electrical stimulation.

The aim of the present study was to investigate the protective effect of integrin ß1 in the treatment of stress urinary incontinence (SUI) by electrical stimulation, and the underlying mechanisms by which electrical stimulation regulates the collagen metabolism of female vaginal wall fibroblasts (FVWFs). FVWFs obtained from the vaginal wall tissue of patients with (Ingelman­Sundberg scale; grade II, n=8; grade III, n=10) or without (n=8) SUI during gynecological operations were isolated by enzymatic digestion and subsequently identified by immunocytochemistry. Following this, cultured FVWFs were treated with an inhibitor of integrin ß1, recombinant human integrin ß1 and electrical stimulation (100 mv/mm, 2 h, 20 Hz), followed by total mRNA and protein extraction. mRNA and protein expression levels of integrin ß1, transforming growth factor (TGF)­ß1 and collagen (COL) I and III in FVWFs were quantified by reverse transcription­quantitative PCR (RT­qPCR) and western blot analysis respectively. Integrin ß1, TGF­ß1 and COL I and III expression levels were decreased in patients with SUI compared with healthy controls, and the grade III group had lower levels than the grade II group. Following electrical stimulation treatment, the expression levels of TGF­ß1, COL I and III were enhanced in the grade II group, but not in the grade III group. Nevertheless, the inhibitor of integrin ß1 reduced the protective effect of electrical stimulation in the grade II group. In addition, electrical stimulation combined with recombinant human integrin ß1 could also protect cells from SUI in the grade III group. The present study provides evidence for the increased degradation of the extracellular matrix and integrin ß1 in the vaginal wall tissues of patients with SUI, and the protective effect of electrical stimulation against SUI via integrin ß1. These results provide a novel mechanism for the treatment of SUI using electrical stimulation.

Role of transforming growth factor ß­1 in the pathogenesis of pelvic organ prolapse: A potential therapeutic target.

The present study aimed to reveal the metabolic alterations of the extracellular matrix (ECM) in uterosacral ligament (USL) with pelvic organ prolapse (POP) and to explore the role of transforming growth factor­ß1 (TGF­ß1) in pathogenesis of POP. For this purpse, 60 participants who underwent hysterectomy for benign indications were enrolled, 30 of which had symptomatic POP (grade II, III or IV) and composed the POP group, and the other 30 had asymptomatic POP (grade I or less) and served as the controls. Collagen fibers, elastin,matrix metalloproteinase (MMP)­2/9, tissue inhibitor of matrix metalloproteinases (TIMP)­2 and TGF­ß1 were examined by Masson's trichrome staining, immunohistochemistry and RT-qPCR using USL biopsies. In vitro, human USL fibroblasts (hUSLFs) were primary cultured, pre-treated with recombinant TGF­ß1 (0, 5, or 10 ng/ml) and then subjected to cyclic mechanical stretching (CMS; 0 or 5,333 µÎµ strain). Changes in the expression levels of collagen type I/III, elastin, TIMP­2, MMP­2/9 and Smad were detected. Our results revealed that at the tissue level, the expression of collagen fibers, elastin, TIMP­2 and TGF­ß1 was significantly reduced in the POP group, while the activities of MMP­2/9 were significantly upregulated, compared with the control group. Statistical analysis indicated that the mRNA expression of TGF­ß1 inversely correlated with the severity of POP partially. Our in vitro experimental data demonstrated that a CMS of 5333 µÎµ strain promoted the degradation of ECM proteins, inhibited the synthesis of TIMP­2, and upregulated the proteolytic activities of MMP­2/9. Pre-treatment with TGF­ß1 attenuated the loss of ECM by stimulating the synthesis of TIMP­2 and inhibiting the activities of MMP­2/9 through the TGF­ß1/Smad3 signaling pathway. On the whole, our data indicate that the reduced anabolism and increased catabolism of ECM proteins in USL are the pathological characteristics of POP. TGF­ß1 not only has a specific value in predicting the severity of POP, but should also be considered as a novel therapeutic target for POP.

Therapeutic Effects of Punicalagin Against Ovarian Carcinoma Cells in Association With ß-Catenin Signaling Inhibition.

AIM: The aim of this study was to investigate the effects of punicalagin, a polyphenol isolated from Punica granatum, on human A2780 ovarian cancer cells in vitro. METHODS: The viability of human A2780 ovarian cells was evaluated using Cell Counting Kit-8 assay. Cell cycle was detected with flow cytometry analysis. The protein expression levels of Bcl-2, Bax, ß-catenin, cyclin D1, survivin, tissue inhibitor of metalloproteinase (TIMP)-2, and TIMP-3 were measured using Western blot analysis. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was determined with gelatin zymography. Wound healing assay was used to determine cell migration. RESULTS: Punicalagin inhibited the cell viability of A2780 cells in a dose- and time-dependent manner, and the cell cycle of A2780 cells was arrested in G1/S phase transition. The treatment also induced apoptosis as shown by the up-regulation of Bax and down-regulation of Bcl-2. On the other hand, punicalagin treatment increased the expressions of TIMP-2 and TIMP-3, decreased the activities of MMP-2 and MMP-9, and inhibited cell migration. In addition, the ß-catenin pathway was suppressed as shown by the down-regulations of ß-catenin and its downstream factors including cyclin D1 and survivin. CONCLUSIONS: Punicalagin may have cancer-chemopreventive as well as cancer-chemotherapeutic effects against human ovarian cancer in humans through the inhibition of ß-catenin signaling pathway.

Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse.

Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four­point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µÎµ, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn­superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports.

MicroRNA-200b suppresses cell invasion and metastasis by inhibiting the epithelial-mesenchymal transition in cervical carcinoma.

The expression of microRNA (miR)-200b is suppressed in numerous tumor types, leading to epithelial-mesenchymal transition, which enables solid tissue epithelial cancers to invade and metastasize. The present study assessed the role of miR-200b in cervical cancer with the aim of clarifying the underlying pathophysiological mechanisms and to identify potential strategies for its prevention and treatment of cervical cancer. Reverse­transcription quantitative PCR revealed that miR­200b was downregulated in invasive cervical carcinoma tissues compared with that in normal adjacent tissues. A Transwell migration assay indicated that transfection of cervical cancer cells with miR­200b mimics significantly inhibited their migratory potential, while migration was enhanced in cells transfected with miR­200b inhibitor. Furthermore, western blot analysis indicated a negative correlation between miR­200b and mesenchymal marker vimentin as well as matrix metalloproteinase­9, which has a key role in tumor invasion and metastasis. In addition, a positive correlation between miR­200b and the epithelial marker E­cadherin was revealed by western blot and immunofluorescence. The results of the present study suggested that miR­200b suppressed the migratory potential of cervical carcinoma cells and therefore their ability to metastasize by inhibiting the epithelial-mesenchymal transition, which may be utilized for the treatment of cervical cancer.

MicroRNA-200b inhibits epithelial-mesenchymal transition and migration of cervical cancer cells by directly targeting RhoE.

Previous studies have identified microRNA-200b (miR-200b) as a powerful regulator of epithelial-mesenchymal transition (EMT) via the control of gene expression. EMT is a critical event that is associated with the initiation of malignant tumor metastasis. A lack of E-cadherin expression and overexpression of vimentin are hallmarks of EMT. It is well­known that RhoE, which is associated with regulation of the actin cytoskeleton and migration via alterations in cell motility, regulates the expression of E-cadherin, matrix metalloproteinase-9 (MMP-9) and vimentin. However, it remains to be elucidated whether miR­200b may alter the molecular behavior of RhoE. The present study aimed to determine whether miR­200b was able to regulate the EMT of cervical cancer, in order to control metastasis. In addition, the correlation between miR­200b and RhoE, E­cadherin and vimentin expression was investigated. Notably, miR­200b was shown to inhibit the function of RhoE and suppress the EMT of cervical cancer. Furthermore, HeLa cells were transfected with miR­200b mimics or inhibitors, and the protein expression levels of E­cadherin, MMP­9, vimentin and RhoE were subsequently detected. A Transwell assay was also conducted, in order to observe the metastatic ability of the HeLa cells. In addition, a luciferase reporter assay was performed using luciferase reporter vectors containing the full length 3'­untranslated region (UTR) of RhoE; miR­200b was able to significantly suppress relative luciferase activity by targeting the 3'­UTR of RhoE. These results suggested that miR­200b may markedly inhibit metastatic potential by regulating cell EMT and inhibiting RhoE; therefore, miR-200b may be considered an effective target for the treatment of patients with highly metastatic cervical cancer.

Collagen metabolic disorder induced by oxidative stress in human uterosacral ligament­derived fibroblasts: A possible pathophysiological mechanism in pelvic organ prolapse.

Pelvic organ prolapse (POP) is a global health problem, for which the pathophysiological mechanism remains to be fully elucidated. The loss of extracellular matrix protein has been considered to be the most important molecular basis facilitating the development of POP. Oxidative stress (OS) is a well­recognized mechanism involved in fiber metabolic disorders. The present study aimed to clarify whether OS exists in the uterosacral ligament (USL) with POP, and to investigate the precise role of OS in collagen metabolism in human USL fibroblasts (hUSLFs). In the present study, 8­hydroxyguanosine (8­OHdG) and 4 hydroxynonenal (4­HNE), as oxidative biomarkers, were examined by immunohistochemistry to evaluate oxidative injury in USL sections in POP (n=20) and non­POP (n=20) groups. The primary cultured hUSLFs were treated with exogenous H2O2 to establish an original OS cell model, in which the expression levels of collagen, type 1, α1 (COL1A1), matrix metalloproteinase (MMP)­2, tissue inhibitor of metalloproteinase (TIMP)­2 and transforming growth factor (TGF)­ß1 were evaluated by western blot and reverse transcription­quantitative polymerase chain reaction analyses. The results showed that the expression levels of 8­OHdG and 4­HNE in the POP group were significantly higher, compared with those in the control group. Collagen metabolism was regulated by H2O2 exposure in a concentration­dependent manner, in which lower concentrations of H2O2 (0.1­0.2 mM) stimulated the anabolism of COL1A1, whereas a higher concentration (0.4 mM) promoted catabolism. The expression levels of MMP­2, TIMP­2 and TGF­ß1 exhibited corresponding changes with the OS levels. These results suggested that OS may be involved in the pathophysiology of POP by contributing to collagen metabolic disorder in a severity­dependent manner in hUSLFs, possibly through the regulation of MMPs, TIMPs and TGF­ß1 indirectly.

Realgar-induced apoptosis of cervical cancer cell line Siha via cytochrome c release and caspase-3 and caspase-9 activation.

OBJECTIVE: To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells. METHODS: The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method. RESULTS: Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar. CONCLUSION: The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

Expression of transforming growth factor-beta 1 and connective tissue growth factor in women with pelvic organ prolapse.

OBJECTIVE: To investigate the presence of transforming growth factor-beta 1 (TGF-ß1) and connective tissue growth factor (CTGF) in women with pelvic organ prolapse (POP). METHODS: This study was conducted from May to December 2009. Fifty patients with POP that underwent vaginal hysterectomy in the Department of Gynecology, Renmin Hospital of Wuhan University, Hubei, Wuhan, China were enrolled in this study. They were divided into: Group 1 (n=10); Group 2 (n=10); and Group 3 (n=10) according to Pelvic Organ Prolapse Quantitation (POP-Q). Meanwhile, 20 cases treated by vaginal hysterectomy for other benign gynecological diseases were selected as the control group. Immunohistochemical staining and Western blot were performed to detect the expression of TGF-ß1 and CTGF. RESULTS: Immunohistochemical staining of TGF-ß1 and CTGF were mainly expressed in the cytoplasm of fibroblast cells. The expression of TGF-ß1 and CTGF protein was significantly negatively correlated with POP-Q stage. There were significantly positive correlations between the expression of TGF-ß1 and CTGF protein. The expression of TGF-ß1 protein among the 3 POP groups were all significantly lower than that of the control group, while there was no significant differences in the expression of TGF-ß1 protein among the POP groups, excluding the comparison between Groups 1 and 3. The expression of CTGF protein in the 3 POP groups were all significantly lower than that of the control group, and significant differences were also detected among the 3 POP groups. CONCLUSION: In this study, we found that the TGF-ß1 and CTGF protein expression may be associated with POP, especially in POP-Q stages.