Cell Cycle Regulatory Proteins Research Articles

Abstract 864: Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2

Abstract Extracted from the root tuber of Ophiopogon japonicas, ophiopogon is well known to have an anti-cancer effect. However, the underlying mechanisms are still largely unknown. Here, we report that Ophiopogon D (OP-D) can inhibit colon cancer cell proliferation and induce apoptosis by inhibiting c-Myc expression through activation of p53 and CNOT2 regulation. Our results showed that OP-D induced p53 expression via ribosomal protein L5 or L11 and inhibited c-Myc expression through CNOT2 in a dose-dependent manner. Additionally, OP-D regulated cyclin D1 and CDK4 which are well known as cell cycle regulatory proteins. Consistently, OP-D inhibited the phosphorylation of AKT expression in a dose-dependent manner. Furthermore, OP-D shortened c-Myc’s half-life in a time-dependent manner. Furthermore, CNOT2 knockdown enhanced the inhibitory effect of OP-D on c-Myc in colon cancer cells. Interestingly, OP-D has increased the apoptotic effect of colon cancer cells when combined with doxorubicin or 5-FU, a treatment already used clinically. Altogether, our results suggested that OP-D regulates colon cancer cell survival and induces apoptosis by inhibiting c-Myc expression via activation of p53 and CNOT2 regulation. Citation Format: Hyun Min Ko, Wona Jee, Do-il Park, Somi Park, Ye-Rin Park, Hyeung-Jin Jang, Ji Hoon Jung. Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 864.

Abstract 691: Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo

Abstract Purpose: Marina crystal minerals (MCM) is a crystallized mixture of minerals and trace elements from sea water. Earlier in vitro studies have shown that MCM possesses apoptotic and immune modulatory effects in human breast cancer cells MDA-MB-231. The current study aimed to evaluate the anticancer effect of MCM in vivo against the growth of murine mammary adenocarcinoma cells and to explore the mechanisms underlying its effect. Materials and Methods: Mice were inoculated intramuscularly with Ehrlich ascites carcinoma (EAC) cells, a breast adenocarcinoma of mouse origin. The tumors grew and became palpable within 9 days post-inoculation. Tumor-bearing mice were injected with MCM intraperitoneally (IP) or intratumorally (IT) at a dose of 40 mg/kg BW for 6 days/week. Different parameters were evaluated, including tumor growth, cell cycle progression, cell cycle regulatory proteins, apoptosis, apoptotic regulatory markers, mitochondrial membrane potential (MMP), natural killer (NK) cell activity, and histopathological effects. Results: Treatment with MCM caused a reduction in tumor volume by 49.4% for IP and 59.5% for IT injection. MCM induced apoptosis in cancer cells, as indicated by the appearance of a sub-G1 peak and confirmed by Annexin V/PI assay and histopathological examination. This was mediated by increased Bax expression, caspase-3 activation, decreased Bcl-2 expression, and MMP disruption. Furthermore, MCM treatment induced G1 cell cycle arrest which was mediated through significantly increased expression of p53, p21, and p27 and decreased expression of Cyclin D1 and PCNA in cancer cells. Finally, MCM treatment markedly enhanced NK cell cytotoxicity. Conclusion: MCM possesses chemopreventive potential to reduce tumor growth by suppressing cell proliferation, inducing apoptosis in EAC cells via a mitochondrial dependent pathway, and activating the immune system. Our results suggest that MCM may have clinical implications for the treatment of breast adenocarcinoma in humans. MCM was provided by the Foundation for Basic Research Institute of Oncology, Japan. Citation Format: Nariman K. Badr El-Din, Mai Alaa El-Dein, Mamdooh Ghoneum. Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 691.

Aberrant Nuclear Translocation of E2F1 and Its Association in Cushing's Disease.

Nonsurgical medical treatments are often performed for Cushing's disease due to high recurrence rates. However, current medical treatment that targets corticotroph adenomas are limited. To develop a treatment that specifically targets corticotrophs in Cushing's disease, it is necessary to identify corticotroph lineage-specific proteins, which are involved in the Cushing's tumor phenotype. We have previously reported that the expression of E2F transcription factor 1 (E2F1), one of the cell cycle regulatory proteins, was increased in corticotrophs in Cushing's disease model mice and was involved in the regulation of POMC gene expression. Phosphorylation of Ser337 of E2F1 (pS337-E2F1) facilitates its binding to the POMC promoter, which was suggested to contribute to elevated POMC expression in corticotrophs. Here, we report that E2F1 expression is specific to the corticotroph lineage in normal human pituitaries and that the E2F1 protein is localized in the cytosol in normal corticotrophs. We show that pS337-E2F1 is localized in the nucleus specifically in Cushing's tumors, while it is localized in the perinuclear cytoplasm in the normal pituitary. This observation demonstrates that pS337 is a marker for Cushing's tumors and suggests that phosphorylation of E2F1 may be a target for developing a novel pharmacological treatment for tumorigenesis and hormone dysregulation of Cushing's disease.

Disruption of cell division prevents gametogenesis in triploid Pacific oysters (Crassostrea gigas)

Triploid oysters with their large size and consistent meat quality have become a major component of the oyster aquaculture industry worldwide. It has been observed that triploid Pacific oysters (Crassostrea gigas) showed high levels of variation and sex-specific differences in sterility. Here, we analyzed mitosis and meiosis during gametogenesis in triploid Pacific oysters and the expression of key cell cycle regulatory proteins, namely members of the cyclin family (A, B, D, E). We found that in female C. gigas, expression of cyclin D and E in gonad of triploids were higher than those in diploids, while expression of cyclin A and B were significantly suppressed in the gonads of triploids. To test whether mitosis of gonia might be arrested at G1/S or S/G2 phase in triploids, we compared EdU labeling in gonadal cells of triploid and diploid oysters. The results showed a similar number of EdU-labelled cells in gonads of triploid and diploid females, indicating that gonia in triploids successfully entered the S-phase as diploids. In male triploids, gonadal expression of the four cyclin genes at the mature stage were similar to that in male diploids at the growth stage with numerous spermatogenic cells. The number of EdU-labelled cells in triploid male gonads at the mature stage was significantly more than that in diploids, suggesting more proliferating cells in triploid gonads in males. Collectively, these data inidate that the sterility of triploid females is likely caused by the failure of germ cells to enter G2-phase during mitosis. In addition, we speculate that gonadal development of triploid males was arrested at the growth stage and defective spermatogenesis might be associated with disruption of meiosis.

P-47 Inhibition of phosphoprotein phosphatase 2A (PP2A) sensitizes pancreatic cancer to PARP inhibitors by modulation of homologous recombination repair

Pancreatic cancer remains one of the most difficult to treat cancers with a 5-years survival of less than 6%, representing one of the most lethal types of cancer, recent research has shown that PP2A inhibitors (LB-100) sensitize pancreatic cancer to chemotherapy and radiation. In addition, the finding from clinical trial study demonstrated that the effectiveness of single-agent Talazoparib for treatment of patients with and without germline BRCA1/2 mutation in ovarian, breast, small cell lung and Pancreatic cancers. The aim of this work was to Ι. investigate activity of LB-100 against pancreatic cancer cells as monotherapy and in combination with PARP inhibitions (Talazoparib). ΙΙ. Investigate the mechanism by which LB-100 and Talazoparib effect pancreatic cancer cells by studying effect of drugs on cell cycle and modulation of cell cycle regulatory proteins and assess the role of treatment combination in control DSB and HR repair through modulation of ATM phosphorylation. Human pancreatic cancer cell lines Panc-1, MIA-Pa-Ca-2 and BxPC-3 were obtained from European Collection of Authenticated Cell Culture (ECACC) and grown in either Dulbecco’s modified Eagle medium (DMEM) (MiaPaCa-2 and Panc-1) or RPMI medium (BxPC-3) with 10% FBS. Cell cultures were maintained in an atmosphere of 5% CO2/95% air at 37 C. PP2A activity was measured by PP2A Immunoprecipitation phosphatase assay Kit (Millipore), cell cytotoxicity were measured by MTT assay, cell cycle determined by flow cytometry. Western blot techniques were used to assess levels of proteins associated with regulation of cell cycle (Cdc2, p-Cdc2, and Cdc25c), apoptosis (caspase3) and DNA damage (γ-H2AX). Data are expressed as the mean ± (SEM) and analysed by an analysis of variance (ANOVA). The results show that LB-100 decreased PP2A activity in all pancreatic cancer cells in dose dependent manner. LB-100 significantly decreased cell viability of Panc-1, MIA-Pa-Ca-2 and BxPC-3 with IC50 (3.94 μM, 6.86 μM, and 10.87 μM) respectively. Interestingly adding 25 nm of Talazoparib further decreased IC50 in all cells (1.88 μM, 5.24 μM and 5.83 μM). Treatment combination attenuated pancreatic cancer cells growth through caspase activation and G2/M cell-cycle arrest. Combination therapy impaired cellular repair and induced DNA double-strand breaks by inducing Υ-H2AX. Our results suggest that treatment combination of LB100 and talazoparib in vitro inhibit cancer cells growth by modulation of the DNA damage response pathway and cell cycle checkpoint abrogation. The combination of PP2A inhibitor with PARP inhibitor has a synergistic effect in vitro. Further in vivo studies are needed to explore this combination as an effective option in the treatment of pancreatic cancer.

Anticancer Activity of Cannabidiol (CBD) in Human Colorectal Cancer Cells: A Mechanistic Study

ObjectivesCannabidiol (CBD) is a major non-psychoactive bioactive component of the phytocannabinoids abundant in cannabis. Its potential use for treatment of colorectal cancer has been proposed by several studies. This study investigated the anti-cancer activity of CBD and the mechanism of that activity in human colorectal cancer cells. MethodsHuman colorectal cancer cell lines (SW620, SW480, HCT116, and Caco-2) and a human normal colon cell line (CCD18CO) were maintained in DMEM supplemented with 10% FBS and antibiotics. Cell viability was measured by MTT assay. Cell cycle phase was measured by flow cytometry. Apoptosis was analyzed by measuring caspase 3/7 enzyme activity. Western blotting was performed to compare expression of proteins regulating cell cycle arrest, apoptosis, and endoplasmic reticulum (ER) stress. Selective inhibitors for cannabinoid receptors were used to test the relevance of each receptor for CBD-mediated cell viability repression and apoptosis induction. Different types of CBD derivatives were tested for effects on cell viability. Data were analyzed using one-way analysis of variance (ANOVA), or two-way ANOVA followed by Bonferroni post-hoc tests (Dunnett T3 tests for unequal variance) for intergroup comparisons. ResultsCBD repressed viability of SW620, SW480, HCT116, and Caco-2 human colorectal cancer cells, with respective IC50 values of 5.4, 10.4, 10.8, and 20 μM for 24-hour treatment and 4.7, 5.8, 5.7, and 13.8 μM for 48-hour treatment. Moreover, CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population with downregulation of cyclin D1, cyclin D3, CDK2, CDK4, and CDK6. It also increased activity of caspase 3/7, production of cleaved PARP, and expression of ER stress proteins (BiP, IRE1α, p-eIF2, ATF3 and ATF4). Repression of cell viability and induction of apoptotic cell death occurred through a mechanism dependent on cannabinoid receptor type 2 (CB2). Suppression of cell viability was also observed in cells treated with other of non-psychoactive cannabinoid derivatives (cannabidivarin, cannabigerol, cannabicyclol, cannabigerovarin). ConclusionsOur data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer. Funding SourcesNo funding source.

Combined Transcriptomic and Proteomic Profiling to Unravel Osimertinib, CARP-1 Functional Mimetic (CFM 4.17) Formulation and Telmisartan Combo Treatment in NSCLC Tumor Xenografts.

The epidermal growth factor receptor (EGFR) is highly expressed in many non-small cell lung cancers (NSCLC), necessitating the use of EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatments. Osimertinib (OSM), a third-generation TKI, is routinely used in clinics, but T790M mutations in exon 20 of the EGFR receptor lead to resistance against OSM, necessitating the development of more effective therapeutics. Telmisartan (TLM), OSM, and cell cycle and apoptosis regulatory protein 1 (CARP-1) functional mimetic treatments (CFM4.17) were evaluated in this study against experimental H1975 tumor xenografts to ascertain their anti-cancer effects. Briefly, tumor growth was studied in H1975 xenografts in athymic nude mice, gene and protein expressions were analyzed using next-generation RNA sequencing, proteomics, RT-PCR, and Western blotting. TLM pre-treatment significantly reduced the tumor burden when combined with CFM-4.17 nanoformulation and OSM combination (TLM_CFM-F_OSM) than their respective single treatments or combination of OSM and TLM with CFM 4.17. Data from RNA sequencing and proteomics revealed that TLM_CFM-F_OSM decreased the expression of Lamin B2, STAT3, SOD, NFKB, MMP-1, TGF beta, Sox-2, and PD-L1 proteins while increasing the expression of AMPK proteins, which was also confirmed by RT-PCR, proteomics, and Western blotting. According to our findings, the TLM_CFM-F_OSM combination has a superior anti-cancer effect in the treatment of NSCLC by affecting multiple resistant markers that regulate mitochondrial homeostasis, inflammation, oxidative stress, and apoptosis.

L-arginine stimulates the proliferation of mouse mammary epithelial cells and the development of mammary gland in pubertal mice by activating the GPRC6A/PI3K/AKT/mTOR signalling pathway.

Amino acids have been shown to affect the development of mammary gland (MG). However, it is unclear whether L-arginine promotes the development of pubertal MG. Therefore, our study aims to explore the effect of L-arginine on the development of MG in pubertal mice. To investigate its internal mechanism of action, we will use mouse mammary epithelial cells (mMECs) line. Whole-mount staining showed that L-arginine can promote the extension of MG duct. In vitro, 0.4 mM L-arginine could activate the G protein-coupled receptor family C, group 6, subtype A (GPRC6A)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway and increase the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4EBP1) to promote the synthesis of cell cycle regulatory protein D1 (Cyclin D1), leading to the dissociation of the retinoblastoma tumour suppressor protein (Rb)-E2F1 transcription factor (E2F1) complex in mMECs and releasing E2F1 to promote cell proliferation. Furthermore, GPRC6A was knocked down or inhibition of the PI3K/AKT/mTOR signalling pathway with corresponding inhibitors completely abolished the arginine-induced promotion of mMECs proliferation. In vivo, it was further confirmed that 0.1% L-arginine can activate the PI3K/AKT/mTOR signalling pathway in the MG of pubertal mice. These results were able to indicate that L-arginine stimulates the development of MG in pubertal mice through the GPRC6A/PI3K/AKT/mTOR signalling pathway.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins’ expression. Additionally, ATM expression suppressed cystathionine β-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

CDC25C is a prognostic biomarker and correlated with mitochondrial homeostasis in pancreatic adenocarcinoma

ABSTRACT Pancreatic adenocarcinoma (PAAD) is a common digestive tract malignant tumor with an extremely poor prognosis. The survival and prognosis may significantly improve if it is diagnosed early. Therefore, identifying biomarkers for early diagnosis is still considered a great clinical challenge in PAAD. Cell Division Cycle 25C (CDC25C), a cardinal cell cycle regulatory protein, directly mediates the G2/M phase and is intimately implicated in tumor development. In the current study, we aim to explore the possible functions of CDC25C and determine the potential role of CDC25C in the early diagnosis and prognosis of PAAD. Expression analysis indicated that CDC25C was overexpressed in PAAD . In addition, survival analysis revealed a strong correlation between the enhanced expression of CDC25C and poor survival in PAAD. Furthermore, pathway analysis showed that CDC25C is related to TP53 signaling pathways, glutathione metabolism, and glycolysis. Mechanically, our in vitro experiments verified that CDC25C was capable of promoting cell viability and proliferation. CDC25C inhibition increases the accumulation of ROS, inhibits mitochondrial respiration, suppresses glycolysis metabolism and reduces GSH levels. To summarize, CDC25C may be involved in energy metabolism by maintaining mitochondrial homeostasis. Our results suggested that CDC25C is a potential biological marker and promising therapeutic target of PAAD.

Methods of senescence induction in human AC16 Cardiomyocytes and associated pathway and phenotypic changes

Cellular senescence, induced by stress factors within a cell, is a state of cell growth arrest that has been found to contribute to age‐related diseases. Senescent cells exhibit a particular phenotype that is generically characterized by increased expression of cell‐cycle regulator proteins, increased expression of senescence associated beta‐galactosidase, and increased oxidative mitochondrial stress. Particularly in the cardiomyocyte, this state of growth arrest contributes to many prevalent cardiomyopathies. However, while it is broadly known that oxidative stress and DNA damage inducing conditions can contribute to a cell becoming senescent, methods to induce senescence in cardiomyocytes effectively have yet to be systematically investigated. In particular, the particular lesions and concentrations that can adequately induce senescence as well as the particular pathway through which senescence is induced in each induction method are unknown. Accordingly, the aim of this study is to optimizethe dosage and regimen of three pro‐senescence insults (hydrogen peroxide, doxorubicin, and UV‐C light) that will best induce senescence as measured by the associated senescent phenotype while gaining deeper understanding of differences in the pathways that are employed within a cell in the process of inducing senescence from different forms of stress. To do so, we used western blots and staining techniques in order to analyze relative expression of classic senescence markers in cells that were exposed to stress conditions as compared to healthy cells. The most relevant proteins that were compared were p16 and p21, cell cycle regulator proteins, and H2A.X, a double‐stranded DNA damage protein. We found that in conditions of 500 μM H2O2, 0.1 μM Dox, and 5 mJ/cm^2 UV‐C light, p21 and H2A.X were significantly upregulated and p16 was interestingly found to be significantly downregulated in all conditions. Treated samples were then filtered and tested via mass spectrometry in order to determine relative protein expressions. Preliminary mass spectrometry data used to compare the different insults showed some evidence of activation of different senescent pathways. Samples insulted with Dox showed an enrichment in the apoptotic pathway while those treated with UV‐C or H2O2 showed enrichment in translation elongation and nonsense mediated decay related pathways. Furthermore, we found that combinatorial treatment of H2O2, UV‐C light and doxorubicin increased expressed mitochondrial oxidative stress as seen by MitoSox staining techniques. In summary, our results showed the different concentrations that can induce senescent phenotypes in cardiomyocytes and the differences in the phenotypes through different senescence induction methods.

Triple Negative Breast Cancer (TNBC) Cells Growth is Inhibited by High Dose Ascorbic Acid

ObjectiveAccording to the World Health Organization report, the incidence of breast cancer mortality is very high worldwide with 2.1 million women are being affected each year and death are estimated at 627,000 women. There are several subtypes of breast cancer. One of these subtypes is known as triple‐negative breast cancer (TNBC). TNBC doesn’t have the receptors for the hormone estrogen, nor progesterone, nor express HER2/neu; therefore, TNBC doesn’t respond to hormonal nor targeted therapies which makes TNBC difficult to treat.The desired outcome of the cancer therapies is to decrease the proliferative activity of the cancer cells, to minimize drug resistance, and to decrease the toxicity of the anticancer drugs. Ascorbic acid has gained popularity as preventive agent for cancer due to its minimal cytotoxic to normal cellular tissue; however, the use of ascorbic acid for the treatment of cancer has controversial history. Taking oral ascorbic acid had no or little effect on cancer treatment, but recent discovery that intravenous ascorbic acid can provide 100‐fold higher plasma levels than oral intake has renewed interest on the role of ascorbic acid in the cancer treatment.HypothesisAt high dose (pharmacological concentrations), ascorbic acid may act as a prodrug to deliver extracellular H2O2 that is selectively toxic to cancer cells, but non‐toxic and well tolerated to normal cells. It is postulated that TNBC cells growth will be inhibited by high dose of ascorbic acid.MethodsAscorbic acid was buffered to pH 7 with sodium hydroxide and prepared immediately before use. MDA‐MB‐231 cells was used as TNBC model. MTT assay, Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Poly (ADP‐Ribose) polymerase (PARP), Casp‐3, Casp‐9, P‐53, Bax, Bcl2, Apaf‐1, MMP2, MMP 9, BRCA1, BRCA2, Bim, Map kinases were conducted to test the hypothesis that ascorbic acid was effective to kill TNBC cells.ConclusionThis finding suggests that high dose ascorbic acid has a potential candidate as effective anti‐cancer for TNBC. At 1.6 mM, ascorbic acid killed MDA‐MB‐231, a TNBC cells by causing increased in caspase 3‐9 activities and by decreasing cell cycle regulatory proteins, Mitogen activated kinase 1‐4, and reactive oxygen species.

CBX3 regulated by miR-139 promotes the development of HCC by regulating cell cycle progression

ABSTRACT Hepatocellular carcinoma (HCC), a major primary liver cancer, is one of the most lethal malignancies worldwide. Increasing evidence has demonstrated that chromobox protein homolog 3 (CBX3) functions as an oncogene in different cancers. However, its expression profiles and biological functions in HCC remain unknown. Data on CBX3 expression in HCC acquired from the GEO and TCGA databases were analyzed. The biological functions of CBX3 in HCC were examined by in vitro experiments. Bioinformatics analysis, qRT-PCR and western blotting were performed to explore the mechanism of CBX3 in HCC. CBX3 mRNA was upregulated in HCC tissues, and overexpression of CBX3 mRNA was negatively correlated with malignancies and poor prognosis in HCC patients. CBX3 knockdown decreased growth, migration and invasion of HCC cells in vitro. Moreover, bioinformatics analysis and experimental observation indicated that CBX3 expression was correlated with cell cycle regulatory proteins in HCC cells. Finally, starBase predicted that miR-139 could directly target CBX3 in HCC. Confirmatory experiments verified that miR-139 overexpression attenuated HCC cell proliferation and migration, and these effects could be reversed by overexpressing CBX3. Our results showed that the miR-139/CBX3 axis may be involved in HCC development by regulating cell cycle progression and may be a promising target in the treatment of HCC.

Anticancer activity of Germacrone terpenoid in human osteosarcoma cells is mediated via autophagy induction, cell cycle disruption, downregulating the cell cycle regulatory protein expressions and cell migration inhibition.

Germacrone a sesquiterpene is a potential pharmacological agent with important medicinal applications. It is a potential anticancer agent and has been reported for anticancer activity against hepatoma cells and breast cancer cells, additionally, it has also shown anti-inflammatory, antioxidant, and antifungal activity. Therefore, this study was designed to testify anticancer activity of germacrone terpenoid in human osteosarcoma cells along with studying its effects of autophagy induction, cell cycle disruption, downregulating the cell cycle regulatory protein expressions and cell migration inhibition. Cell proliferation rate was examined by MTT assay and phase contrast inverted microscopy was performed for morphological analysis. Further, flowcytometry was implemented to examine different cell cycle phases. Transwell assay was executed for the monitoring of cell migratory tendency of osteosarcoma cells. Finally, the levels of pro-autophagic and cell cycle allied proteins were checked by Western blot analysis. MTT assay results designated potential inhibition of osteosarcoma cell viability by germacrone drug in a dose and time-reliant manner. Further, phase contrast inverted microscopy depicted significant morphological changes in osteosarcoma cells after germacrone exposure, which were indicative of autophagic cell death. Next, transmission electron microscopy evaluated the formation of autophagic vesicles which are the trademark for autophagy. The autophagy allied protein expressions were observed through Western blotting indicating enhanced levels of pro-autophagic proteins (Becalin-1, LC3-I and -II). Hence, it may be depicted that the anti-proliferation effects of germacrone may be of autophagy inducing potential. Next, flowcytometric analysis revealed the cell cycle inhibitory effects of germacrone in osteosarcoma cells and the results indicated cell cycle arrest at S-phase. Cell cycle allied protein levels indicated declination in their expressions after germacrone exposure. Finally, transwell assay specified inhibitory effects on cell migration of osteosarcoma cells by germacrone in a dose-reliant manner. In conclusion, the results of the present investigation specified that germacrone drug is a potential anticancer agent against osteosarcoma cells. The anticancer effects were found to be mediated via autophagy induction, cell cycle disruption, downregulating the cell cycle regulatory protein expressions, and cell migration inhibition.

Cobalt (III) complex exerts anti-cancer effects on T cell lymphoma through induction of cell cycle arrest and promotion of apoptosis.

Cobalt-based compounds are emerging as a non-platinum-based anti-cancer effective therapeutic agent. However, there is a limited study regarding the therapeutic efficacy of Cobalt-based drugs against Non-Hodgkin's Lymphoma (NHLs) such as T cell lymphoma. Therefore, in the present study we investigated the anti-tumor role of cobalt(III) complex [Co(ptsm)NH3(o-phen)]·CH3OH on Dalton's Lymphoma (DL) cells. Cytotoxicity of the cobalt complex was estimated by MTT assay. Analysis of mitochondrial membrane potential, cell cycle and Reactive oxygen species (ROS) generation, and Annexin V/PI staining was done by Flow cytometry, while AO/EtBr staining by fluorescence microscopy in cobalt complex treated DL cell. Expression of cell cycle and apoptosis regulatory protein was analyzed by Western blotting. In addition, in vivo study of the cobalt complex was evaluated in well-established DL bearing mice by monitoring physiological parameters and mean survival time. Our study showed that cobalt complex triggered apoptosis and induced cell cycle arrest in DL cells. Furthermore, this also decreased mitochondrial membrane potential and increased intracellular ROS generation in cancer cells. In addition, changed expression of cell cycle and apoptosis regulatory protein was found with enhanced activity of caspase-3 and 9 in the treated cells. Additionally, administration of cobalt complex showed a significant increase in the survivability of tumor-bearing host, which was accomplished by decreasing physiological parameters. Taken together, these data revealed anti-tumor potential of cobalt complex against DL cells through cell cycle arrest and mitochondrial-dependent apoptosis. Henceforth, cobalt-based drugs could be a new generation therapeutic drug to treat hematological malignancies.

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells.

Background: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. Methods: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. Results: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. Conclusions: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

A review on the genetic polymorphisms and susceptibility of cancer patients in Bangladesh.

Cancer is one of the major health burdens worldwide, and genetic polymorphisms in individuals are closely associated with cancer susceptibility. Like in many other developing countries, the risk of cancer is increasing among Bangladeshi population. Genetic polymorphisms in xenobiotic metabolic enzymes (CYP1A1, CYP2A6, CYP3A4, CYP3A5, NAT2, SULT1A), cell cycle regulatory proteins (TP53, HER2, MDM2, miR-218-2, TGFB), cell signaling protein (CDH1), DNA repair proteins (BRCA1, BRCA2, EXO1, RAD51, XRCC2, ECCR1, ERCC4, XPC, ERCC2), and others (HLA-DRB1, INSIG2, GCNT1P5) have been found to be associated with various cancers like cancers of breast, bladder, cervix, colon, lung, prostate, etc. in different studies with Bangladeshi population. In this review article, we have discussed these gene polymorphisms associated with cancers in the Bangladeshi population, and also made a comparison with other ethnic groups. This will probably be helpful in understanding drug effects, drug resistance, and personalized medicine in the population of this region.

Circular RNA RHOT1 Regulates miR-142-5p/CCND1 to Participate in Chondrocyte Autophagy and Proliferation in Osteoarthritis

Background Osteoarthritis (OA) serves as one of the most prevalent types of joint disorders and is a leading cause of symptoms of stiffness, swelling, and arthralgia. Circular RNAs (circRNAs) have been reported to participate in various cellular processes by competing with microRNAs. Meanwhile, cyclinD1 (CCND1) is a key cell cycle regulatory protein and plays a crucial role in OA progression. Nevertheless, the function of circRHOT1 in the modulation of OA progression is still obscure. Here, we explored the effect of circRHOT1 on autophagy and extracellular matrix (ECM) in OA. Methods The expression of circRHOT1 and autophagy markers was detected in OA samples. The effect of circRHOT1 on OA was analyzed in the OA rat model. The function of circRHOT1 in the regulation of OA was assessed by CCK-8, colony formation, flow cytometry analysis, quantitative real-time PCR, Western blot analysis, and luciferase reporter gene assay in chondrocytes. Results We observed that autophagy markers, including LC3 and beclin1, were repressed in clinical OA samples. The expression of circRHOT1 and CCND1 was induced but the miR-142-5p expression was reduced in clinical OA samples. The miR-142-5p expression was negatively correlated with circRHOT1 and CCND1, and the circRHOT1 expression was positively associated with CCND1 in clinical OA samples. Meanwhile, the apoptosis was induced in OA rats but the depletion of circRHOT1 could block the phenotype in the rats. The articular cartilage degeneration was promoted in OA rats, while the knockdown of circRHOT1 repressed the degeneration. The serum levels of CTX-II and COMP were increased in OA rats, and the silencing of circRHOT1 downregulated levels of these proteins. In addition, the expression of collagen II and aggrecan was promoted by the depletion of circRHOT1 in the OA rats. Significantly, the expression of LC3 and beclin1 was suppressed in OA rats, in which the knockdown of circRHOT1 could reverse the effect. Moreover, the depletion of circRHOT1 repressed the cell viability and proliferation but induced apoptosis of chondrocytes. The expression levels of LC3, beclin1, collagen II, and aggrecan were induced by circRHOT1 knockdown. Mechanically, circRHOT1 was able to enhance the CCND1 expression by sponging miR-142-5p in chondrocytes. The overexpression of CCND1 or the inhibition of miR-142-5p reversed circRHOT1 depletion-mediated chondrocyte phenotypes. Conclusions Circular RNA RHOT1 enhances the CCND1 expression by sponging miR-142-5p to inhibit chondrocyte autophagy and promote chondrocyte proliferation in osteoarthritis. Our findings provided a promising therapeutic target for OA.

Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration.

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.

Effects of Icodextrin Solution (Adept®) on Ovarian Cancer Cell Proliferation in an In Vitro Model.

Background and objective: Anti-adhesion barriers are currently used during ovarian cancer surgery to decrease adhesion-related morbidity. Adept® (4% icodextrin) solution, a liquid anti-adhesion material, has been widely used during gynecologic surgeries, though the risk of this barrier for oncologic surgery is controversial. The aim of this study was to determine the effect of Adept® solution on the proliferation of ovarian cancer cells. Materials and methods: We assessed the dose- and time-dependent effects of icodextrin on the growth and proliferation of OVCAR-3 and A2780 human ovarian tumor cell lines in vitro. Cell growth was determined by cell number counting. Expressions of cell cycle-regulation proteins (cyclin D1 and cyclin B1) were determined using Western blot analysis. Results: Adept® did not significantly increase ovarian cancer cell growth when tested at various concentrations (0, 1, 5, 10, 15, and 20%, equal to 0, 0.04, 0.2, 0.4, 0.6 and 0.8% icodextrin) and different time points (1–3 days) compared to control cells. Moreover, the protein levels of cyclin D1 and B1 were not overexpression-elevated in icodextrin-treated ovarian cancer cells, either with an increasing concentration or with an increasing treated time. These results demonstrated that Adept® does not activate the growth or proliferation of ovarian cancer cells in either a dose- or time-dependent manner. Conclusions: This study supports the use of Adept® solution as a safe anti-adhesion barrier for ovarian cancer surgery, though further in vivo studies are necessary.