Cyclin CDK4 Research Articles

Abstract 5358: Involvement of solute carrier transporters SLC25A17 and SLC27A6 in metabolic reprograming of castration-resistant prostate cancer

Abstract Enzalutamide (XTANDI®), an antiandrogen, is used for the treatment of advance-stage prostatecancer. Approximately, 60% of patients receiving enzalutamide show initial remission followedby disease relapse with the emergence of highly aggressive castration-resistant prostate cancer.Solute carrier (SLC) transporters play a critical role in cancer drug resistance by altering cellularmetabolism. We have recently reported the predominance of SLC25A17 and SLC27A6 duringenzalutamide resistance (Cells 9(12):2535, 2020) by generating enzalutamide resistant (C4-2B-Enzu) cells exposing human prostate cancer C4-2B cells in the presence of increasingconcentration of enzalutamide (1-20 µM) and maintained in the same condition for six months.However, the role of these molecules during metabolic reprogramming have not beenelucidated. Knockdown of SLC25A17 and SLC27A6 gene by sgRNA transfection suppressedcell proliferation, and migration in C4-2B-Enzu cells. Analysis of cell cycle showed that in theabsence of SLC25A17 and SLC27A6 there is induction of G1/S cell cycle arrest and reductionin the protein expression of checkpoint factors viz. Cyclin D1, CDK6, CDK4, and CDK2.SLC25A17 and SLC27A6 knockdown resulted in downregulation of acetyl-CoA carboxylase(ACC), fatty acid synthase (FASN) and Acyl-CoA synthetase long chain family member 5(ACSL5) with simultaneous decrease in the levels of lactic acid, triglyceride and citric acid in C4-2B-Enzu cells. In addition, SLC25A17 knockdown resulted in marked increase in apoptosis asevident by increase in protein expression of RIPK3 and BAX, compared to SLC27A6 knockdownin C4-2B-Enzu cells. Taken together, our results demonstrate that solute carriers SLC25A17and SLC27A6 play an important role in the development of enzalutamide resistance throughmetabolic reprogramming and loss of these molecules results in cell death. Thus, SLC25A17and SLC27A6 may serve as a therapeutic target for enzalutamide resistant prostate cancer. Citation Format: Prem P. Kushwaha, Shiv S. Verma, Eswar Shankar, Spencer Lin, Sanjay Gupta. Involvement of solute carrier transporters SLC25A17 and SLC27A6 in metabolic reprograming of castration-resistant prostate cancer [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 5358.

Abstract 6261A: Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa modulate apoptosis via induction of pro-apoptotic and anti-apoptotic proteins in MDA-MB-157 and 93B breast cancer cells

Abstract Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in Traditional Chinese Medicine for treating colon cancer, glioblastoma, and ovarian cancer liver, lung, and rectal cancers. These two herbs are included in most of the herbal cancer treatment formulas in China and Taiwan hospitals. We previously demonstrated that aqueous extracts of SB and OD inhibited mutagenesis, DNA binding, and metabolism of aflatoxin B1 and benzo[a]pyrene in carcinogenesis. In studies by Hugo (2006) and Perez (2010), the efficacy of BZL 101 (an extract of SB) has been demonstrated in preliminary Phase Ia and Ib clinical trials in metastatic breast cancer patients. It is also shown to induce an S phase arrest of the cell cycle by affecting the Cycline A2 and CDK2 expression in estrogen-sensitive MCF7 and estrogen insensitive MDA-MB-231 breast cancer cells. MeOH and BuOH extract of OD have been illustrated to reduce MCF7 invasion via the inhibition of MMP-9 and ICAM-1 apoptotic proteins expression. Ursolic acid of OD could inhibit breast cancer metastasis by suppressing glycolytic metabolism through activating SP1/Caveolin-1 signaling. In this study, the effectiveness of aqueous extract of SB and OD in the modulation of apoptosis of breast cancer cell lines MDA-MB-157 and 93B (a drug-resistant APC-mutant of it) were investigated. Assessments were performed using green/red/blue fluorescent Apoptosis/Necrosis Detection Test and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that 3-hours treatment with 2.5 mg aqueous extract of SB induced a statistically significant percentage of apoptosis in MDA-MB-157 (46.5 ± 7.5% > 16%± 1.0%, p < 0.05) and 93B (46.5 ± 7.5% > 16%± 1.0%, p < 0.05) as compared to the negative control. Similar results were obtained with the OD treatment. From the antibody array-membrane experiments, modulation of various apoptosis markers by SB such as the pro-apoptotic proteins BID and BIM (p=0.019; p=0.007) in MDA-MB-157 and Bad (p=0.002), Bax (p=0.020), BID (p=0.001), p27 (p=0.004), p53 (p=0.022) in 93B as compared to the negative controls; and anti-apoptotic markers BcL-2, FasL, IGF-I, and p21 were also observed. OD treatment also induced similar significant production of these two types of proteins. These results suggest that SB and OD contain phytochemicals that induce apoptosis in MDA-MB-157 and 93B via the modulation of these pro-apoptotic and anti-apoptotic proteins. Citation Format: MinSeo Kang, Nathaly Manrique, Rekha Issac, Sarah Wolf, Jasmine Cha, SungBeen Han, Hugo Jie Qin, Lun Kim, Padma P. Uppala, Brian Yuen Yau Wong. Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa modulate apoptosis via induction of pro-apoptotic and anti-apoptotic proteins in MDA-MB-157 and 93B breast cancer cells [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 6261A.

Effects of calcitriol on the cell cycle of rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is a type of cancer of skeletal muscle. Calcitriol is the active form of vitamin D3, also recognised as a steroid hormone called 1α, 25-dihydroxy vitamin D3 (1,25D). We previously reported that 1,25D promoted cell proliferation and differentiation in non-cancerous skeletal muscle cells C2C12. The aim of this work is to evaluate some of the events triggered by 1,25D in RD cells, a human RMS cell line. In this work we reported that RD cells expressed vitamin D receptor (VDR) and treatment with 1,25D reduced VDR expression at 72 h. At the same time an acute decrease in viable cells as well as in cells in S-phase of cell cycle was also observed. Furthermore, up-regulation of p15INK4b was accompanied in a timely manner by down-regulation of cyclin D3, p21Waf1/Cip1 and myogenin protein levels. Simultaneously, 1,25D induced early apoptosis markers such as cyclin D1 and CDK4, and the disruption of the mitochondrial network together with a redistribution of mitochondria around the nucleus. Finally, 1,25D induced changes in the plasma membrane of RD cells associated with early and late apoptosis at 72 h, as determined by flow cytometry. Taken together, these results determine that treatment with 1,25D for 72 h triggers apoptosis in RD cells.

Phase 2 study to evaluate palbociclib in combination with irinotecan and temozolomide in pediatric patients with recurrent or refractory Ewing sarcoma.

TPS11583 Background: Palbociclib (PD-0332991) is a highly selective, reversible, small molecule inhibitor of cyclin-dependent kinases (CDK) 4 and 6, administered orally. Functional dependence of Ewing Sarcoma (EWS) cell lines and tumor xenografts on Cyclin D1/CDK4 via genetic knockdown has shown both Cyclin D1 and CDK4 as critical dependencies for EWS cell line proliferation. The phase 1 portion of the study established the recommended Phase 2 dose (RP2D) with two chemotherapy backbones in children with solid tumors. The Phase 2 portion of the study now enrolls patients with EWS. The primary objective of this portion of the study is to determine whether the addition of palbociclib to irinotecan (IRN) and temozolomide (TMZ) will prolong event-free survival (EFS) of pediatric and young adult patients with recurrent or refractory EWS. Methods: Patients with recurrent or refractory EWS are randomized 2:1 to receive either palbociclib in combination with IRN and TMZ or IRN and TMZ alone. Randomization is stratified by type and time of disease recurrence (primary refractory or 1st recurrence <2 years vs. 1st recurrence ≥2 years or 2nd or greater recurrence). The primary efficacy endpoint is EFS per investigator assessment. Secondary efficacy endpoints include objective response, progression-free survival and overall survival. An interim futility analysis will be conducted to allow for early stopping of the study due to futility/no signal of activity based on the primary endpoint of EFS. Safety and planned interim efficacy data will be assessed by an Independent Data Monitoring Committee (DMC). Key eligibility criteria include: recurrent or refractory EWS with evaluable disease, no known bone marrow metastases, histopathological confirmation of EWSR1-ETS or FUS-ETS rearrangement or availability of formalin fixed paraffin embedded (FFPE) tumor tissue sample for central testing, age ≥2 and <21 years at the time of study entry. Treatment intervention: Patients randomized to palbociclib with IRN and TMZ treatment arm will receive palbociclib at the RP2D of 75 mg/m2 orally (either as a capsule or oral solution) once daily on Days 1-14 of each 21-day treatment cycle. TMZ will be administered orally once daily at 100 mg/m2 on Days 1-5 (intravenously (IV) if patient cannot swallow the TMZ capsule). IRN will be administered IV at 50 mg/m2 on Days 1-5. Patients randomized to the chemotherapy only treatment arm will receive IRN and TMZ at the same doses on Days 1-5 of the 21-day treatment cycle. Treatment will continue until disease progression, patient and/or legal guardian refusal, unacceptable toxicity , or up to 24 months of treatment, whichever occurs first. The Phase 2 enrolment has been initiated and 1/75 patients has been enrolled as of Jan 2022. Clinical trial information: NCT03709680.

Sirt2 positively regulates muscle regeneration after Notexin-induced muscle injury

Sirt2 regulates various biological processes by deacetylating target genes. Despite roles in regulating proliferation, cell cycle, and glucose metabolism, which are closely associated with skeletal muscle physiology, Sirt2 functions in this tissue remain unclear. In this study, genetic deletion of Sirt2 delayed muscle regeneration after Notexin-induced muscle injury. Gene expressions of myogenic regulatory factors, including Myf5, MyoD, and Myogenin, and cell cycle regulators, such as cyclin D1 and CDK2, were repressed in Sirt2 knockout mice after injury. Also, Sirt2 knockout mice presented muscle atrophy after muscle injury which is associated with the down-regulation of anabolic signaling and the up-regulation of catabolic signaling, in particular, increased atrogin1 transcriptional expression. Thus, Sirt2 positively regulated skeletal muscle regeneration after muscle injury by regulating transcriptional expression involved in myogenesis, cell cycle, and anabolic and catabolic signaling. Based on the in vivo analyses, Sirt2 could function as an interventional therapeutic for chronic myopathy, which is characterized by impaired muscle regeneration and muscle atrophy.

Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells.

Simple SummaryHepatocellular carcinoma (HCC) progression depends on two major processes, tumor growth and invasion. The present study investigated how these events are linked. A panel of HCC cell lines were stimulated with insulin-like growth factor-1 (IGF1) and the biological behavior was evaluated. IGF1 activated the proliferation and invasion cascade by altering the expression level of integrin α subtypes, which were associated with the AKT-mTOR pathway and the CDK-Cyclin axis. We assume that HCC progression is controlled by a fine-tuned network between IGF1 driven integrin signaling, the Akt-mTOR pathway, and the CDK-Cyclin axis. Concerted targeting of these pathways may, therefore, become an innovative option to prevent cancer dissemination.Integrin receptors contribute to hepatocellular carcinoma (HCC) invasion, while AKT-mTOR signaling controls mitosis. The present study was designed to explore the links between integrins and the AKT-mTOR pathway and the CDK-Cyclin axis. HCC cell lines (HepG2, Huh7, Hep3B) were stimulated with soluble collagen or Matrigel to activate integrins, or with insulin-like growth factor 1 (IGF1) to activate AKT-mTOR. HCC growth, proliferation, adhesion, and chemotaxis were evaluated. AKT/mTOR-related proteins, proteins of the CDK-Cyclin axis, focal adhesion kinase (FAK), and integrin-linked kinase (ILK) were determined following IGF1-stimulation or integrin knockdown. Stimulation with collagen or Matrigel increased tumor cell growth and proliferation. This was associated with significant alteration of the integrins α2, αV, and β1. Blockade of these integrins led to cell cycle arrest in G2/M and diminished the number of tumor cell clones. Knocking down the integrins α2 or β1 suppressed ILK, reduced FAK-phosphorylation and diminished AKT/mTOR, as well as the proteins of the CDK-Cyclin axis. Activating the cells with IGF1 enhanced the expression of the integrins α2, αV, β1, activated FAK, and increased tumor cell adhesion and chemotaxis. Blocking the AKT pathway canceled the enhancing effect of IGF on the integrins α2 and β1. These findings reveal that HCC growth, proliferation, and invasion are controlled by a fine-tuned network between α2/β1-FAK signaling, the AKT-mTOR pathway, and the CDK–Cyclin axis. Concerted blockade of the integrin α2/β1 complex along with AKT-mTOR signaling could, therefore, provide an option to prevent progressive dissemination of HCC.

Structural Characterization and Anti-breast Cancer Activity in vitro of a Novel Polysaccharide From Cymbopogon citratus.

Cymbopogon citratus is an important functional food, widely used for flavoring in Africa and South America. In this study, a novel high-molecular-weight polysaccharide (CCP) from C. citratus was extracted, and its structural characteristics and anti-breast cancer activity in vitro were investigated. CCP contained both α and β configurations and mainly composed of galactose (36.89%), arabinose (23.97%), glucose (18.35%) and rhamnose (9.36%) with an average molecular weight of 1.98 × 106 Da. The main glycosyl residues of CCP detected by methylation analysis were 1,3,6-linked Galp, 1,3-linked Glcp, 1,5-linked Araf , T-Araf , and T-Rhap. In vitro experiments suggested that CCP significantly inhibited the proliferation of MDA-MB-231 cells, decreased the expressions of cyclin D1 and CDK4 and stocked cells at G0/G1 phase. Meanwhile, the typical morphological features of apoptotic cells were also observed. Combining with the consequences of Annexin V-FITC/PI staining, Hoechst 33258 staining and western blot analysis, CCP induced apoptosis of MDA-MB-231 cells by triggering the Fas/FasL-mediated death receptor pathway. Overall, these results provide a theoretical basis for the application of C. citratus polysaccharide as a potential anti-breast cancer agent in functional food and medicine.

Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol.

Ailanthoidol (ATD) has been isolated from the barks of Zanthoxylum ailanthoides and displays anti-inflammatory, antioxidant, antiadipogenic, and antitumor promotion activities. Recently, we found that ATD suppressed TGF-β1-induced migration and invasion of HepG2 cells. In this report, we found that ATD exhibited more potent cytotoxicity in Huh7 hepatoma cells (mutant p53: Y220C) than in HepG2 cells (wild-type p53). A trypan blue dye exclusion assay and colony assay showed ATD inhibited the growth of Huh7 cells. ATD also induced G1 arrest and reduced the expression of cyclin D1 and CDK2. Flow cytometry analysis with Annexin-V/PI staining demonstrated that ATD induced significant apoptosis in Huh7 cells. Moreover, ATD increased the expression of cleaved PARP and Bax and decreased the expression of procaspase 3/8 and Bcl-xL/Bcl-2. In addition, ATD decreased the expression of mutant p53 protein (mutp53), which is associated with cell proliferation with the exploration of p53 siRNA transfection. Furthermore, ATD suppressed the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and the expression of mevalonate kinase (MVK). Consistent with ATD, the administration of S3I201 (STAT 3 inhibitor) reduced the expression of Bcl-2/Bcl-xL, cyclin D1, mutp53, and MVK. These results demonstrated ATD’s selectivity against mutp53 hepatoma cells involving the downregulation of mutp53 and inactivation of STAT3.

The Effect of Nano Albumin Combined with Paclitaxel on Drug Resistance of Breast Cancer Through Regulating ATP Binding Cassette Subfamily B Member 1 (ABCB1)

The paclitaxel is a common-used chemotherapy drug and its combination with nano albumin reduces drug side effect. However, whether nab-paclitaxel affects drug resistance of breast cancer remains unclear. This study intends to discuss the mechanism of drug resistance induced by nab-paclitaxel. The drug resistance of MCF-7/nab-paclitaxel in MCF-7 cell and cell proliferation was detected by MTT along with analysis of ABCB1 expression, cell cycle, and apoptosis. There was stronger drug resistance of nab-paclitaxel in the MCF-7/nab-paclitaxel cell group through be adopted with different concentration of nab-paclitaxel at the 0th hour, 24th hour and 48th hour. There was remarkable abnormal expression of the ABCB1 in the MCF-7/nab-paclitaxel cell group. The si-ABCB1 could release the quantity of the MCF-7/nab-paclitaxel cell blocked at S period. And the si-ABCB1 could reduce the expression of cyclin D1 and CDK2 in the MCF-7/nab-paclitaxel cell notably. But the expression level of p21 was increased when there was high concentration of si-ABCB1. The si-ABCB1 could increase the quantity of the MCF-7/nab-paclitaxel cell at the later period of cell apoptosis notably. The rat’s tumor growth was delayed obviously at the MCF-7/nabpaclitaxel cell group treated by si-ABCB1. But the inhibiting effect of the MCF-7/nab-paclitaxel cell on tumor growth was less. There was stronger drug resistance of cell for the nano albumin combined with paclitaxel. The function of cell proliferation in breast cancer was restrained by the nano albumin combined with paclitaxel mainly through inducing the expression of ABCB1, adjusting the growth of cell cycle and the expression of P21/BCL-2 protein.

CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

The disassembly of integrin-containing focal adhesions (FAs) at mitotic entry is essential for cell rounding, mitotic retraction fibre formation, bipolar spindle positioning and chromosome segregation. The mechanism that drives FA disassembly at mitotic entry is unknown. Here, we show that the CDK1–cyclin B1 complex phosphorylates the integrin activator kindlin, which results in the recruitment of the cullin 9–FBXL10 ubiquitin ligase complex that mediates kindlin ubiquitination and degradation. This molecular pathway is essential for FA disassembly and cell rounding, as phospho-inhibitory mutations of the CDK1 motif prevent kindlin degradation, FA disassembly and mitotic cell rounding. Conversely, phospho-mimetic mutations promote kindlin degradation in interphase, accelerate mitotic cell rounding and impair mitotic retraction fibre formation. Despite the opposing effects on kindlin stability, both types of mutations cause severe mitotic spindle defects, apoptosis and aneuploidy. Thus, the exquisite regulation of kindlin levels at mitotic entry is essential for cells to progress accurately through mitosis.

Mitochondrial micropeptide STMP1 promotes G1/S transition by enhancing mitochondrial complex IV activity

The roles of micropeptides in cell cycle regulation and cancer development remain largely unknown. Here we found that a micropeptide STMP1 (small transmembrane protein 1) was up-regulated in multiple malignancies including hepatocellular carcinoma (HCC), and its high level was associated with short recurrence-free survival of HCC patients. Gain- and loss-of-function analyses revealed that STMP1 accelerated cell proliferation and clonogenicity invitro and tumor growth invivo, and silencing STMP1 blocked G1/S transition. Mechanistically, STMP1 promoted the mRNA and protein levels of CCNE2, CDK2, and E2F1. STMP1 was localized in the inner membrane of mitochondria and interacted with mitochondrial complex IV and then enhanced its activity. Moreover, treatment with the mitochondrial complex IV inhibitor tetrathiomolybdate dramatically abrogated the promoting effect of STMP1 on cell proliferation and the expression of cyclin E2, CDK2, and E2F1. These results suggest that STMP1 may promote G1/S transition and cell proliferation by enhancing mitochondrial complex IV activity, which highlights STMP1 as a new regulator of the cell cycle and a potential target for anti-cancer therapy.

Pyrogallol-Phloroglucinol-6, 6-Bieckol Restored Primary Cilia Length, Which Was Decreased by High-Fat Diet in Visceral Adipose Tissue, and Decreased Adipogenesis

Length of primary cilia, which involves cell cycle reentry and disassembly of cilia, promotes cell mitosis. It is known that the cilia length in adipose tissue of the high-fat diet (HFD) animals was shortened and accompanied by increased adipogenesis. Male C57BL/6N mice were randomly divided into groups. The mice group was given the normal fat diet (NFD/saline), HFD mice group for 4 weeks, and then HFD was also treated for the next 4 weeks with saline (HFD/saline), Ecklonia cava extract (HFD/ECE), or pyrogallol-phloroglucinol-6, 6-bieckol, a segment of ECE (HFD/PPB). We evaluated the effect of ECE and PPB on modulating cilia length of visceral adipose tissue and decreasing adipogenesis by decreasing cell cycle reentry using an HFD-fed mouse model. ECE and PPB decreased physiological changes, which increased by HFD, but ECE and PPB decreased the upregulation of the IL-6/STAT3/AURKA signaling pathway, which is involved in cilia disassembly. In addition, ECE or PPB elongated the cilia and decreased cyclin A2 and Cdk2 expression, which promote cell cycle reentry, and decreased the adipogenesis genes. PPB and ECE restored cilia length and decreased adipogenesis through modulating the IL-6/STAT3/AURKA pathway and decreasing cell cycle reentry in the visceral adipose tissue of HFD/saline mice group.

Cyclin J-CDK complexes limit innate immune responses by reducing proinflammatory changes in macrophage metabolism.

Toll-like receptor (TLR) stimulation induces glycolysis and the production of mitochondrial reactive oxygen species (ROS), both of which are critical for inflammatory responses in macrophages. Here, we demonstrated that cyclin J, a TLR-inducible member of the cyclin family, reduced cytokine production in macrophages by coordinately controlling glycolysis and mitochondrial functions. Cyclin J interacted with cyclin-dependent kinases (CDKs), which increased the phosphorylation of a subset of CDK substrates, including the transcription factor FoxK1 and the GTPase Drp1. Cyclin J-dependent phosphorylation of FoxK1 decreased the transcription of glycolytic genes and Hif-1α activation, whereas hyperactivation of Drp1 by cyclin J-dependent phosphorylation promoted mitochondrial fragmentation and impaired the production of mitochondrial ROS. In mice, cyclin J in macrophages limited the growth of tumor xenografts and protected against LPS-induced shock but increased the susceptibility to bacterial infection. Collectively, our findings indicate that cyclin J-CDK signaling promotes antitumor immunity and the resolution of inflammation by opposing the metabolic changes that drive inflammatory responses in macrophages.

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

Altersolanol B, a fungal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adenocarcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways

The present study focused on the apoptosis-inducing effects and cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line, MCF-7. AB demonstrated approximately 4-fold greater antiproliferative activity in ER+ MCF-7 cells (IC50 5.5 μM) compared to the ER-negative (triple-negative) MDA-MB-231 (IC50 21.3 μM). The viability of normal breast fibrocystic epithelial cells, MCF-10A, was unaffected. AB induced intrinsic apoptosis in MCF-7 cells; it triggered the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP), upregulated the expression of pro-apoptotic Bax, and downregulated the expression of anti-apoptotic Bcl-2. AB induced cell cycle arrest at G0/G1, as indicated by the downregulation of key checkpoint proteins operating at the G0/G1 phase of the cell cycle (cyclin D1, CDK4 and CDK2). The observed increase in p21Waf1/Cip1 and p53 expression may facilitate cell cycle arrest, and the subsequent induction of apoptosis. AB lacked significant effects on intracellular ROS levels, while it down-regulated nuclear factor erythroid 2-related factor 2 (Nrf2), and the Nrf2-dependent antioxidant enzyme, heme oxygenase-1. The compound disrupted AKT signaling through the downregulation of phospho-AKT and phospho-FOXO1, and the upregulation of PTEN, a phosphatase and tumor suppressor that negatively regulates the PI3K/AKT pathway. AB also disrupted the phosphorylation of AKT-controlled eukaryotic translation initiation factor, 4E-BP1, and GSK-3β, both of which are aberrantly regulated in human cancer. The AB-dependent downregulation of NF-κB was corroborated by the inhibition of TNFα-induced NF-κB activity as monitored in a luciferase reporter. The NF-κB inhibitory activity of AB was 3-fold more potent than that of the standard inhibitor, N-p-Tosyl-l-phenylalanine chloromethyl ketone. In addition to reducing the pro-survival effects of NF-кB, the inhibition of AKT phosphorylation by AB may also lead to FOXO1-mediated growth arrest and apoptosis. AB upregulated the expression of phospho-MKK4 and phospho-p38, and downregulated the expression of phospho-MEK1/2 and phospho-ERK1/2 indicating opposing effects on the two important oncogenic signaling cascades that are aberrantly activated in many cancers. AB disrupted both the AKT and ERK1/2 signaling pathways leading to apoptosis in ER+ MCF-7 cells through mitochondria-associated mechanisms coupled with the potent inhibition of NF-кB activation. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. Our preliminary study suggested that the THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthraquinone that possess intrinsic redox activity and DNA interacting ability. This study supports the continued investigation of THAQs as lead molecules in anticancer drug discovery and development.

Positive regulation of ataxia-telangiectasia-mutated protein (ATM) by E2F transcription Factor 1 (E2F-1) in cisplatin-resistant nasopharyngeal carcinoma cells

ObjectiveTo explore the mechanism of E2F transcription Factor 1 (E2F-1)-mediated ataxia-telangiectasia-mutated protein (ATM) in cisplatin (DDP)-resistant nasopharyngeal carcinoma (NPC).MethodsE2F-1 and ATM expression was assessed in DDP-resistant NPC cell lines (CNE2/DDP and HNE1/DDP) and parental cells. Then, DDP-resistant NPC cells were transfected with control shRNA (short hairpin RNA) or E2F-1 shRNAs with or without ATM lentiviral activation particles. The half maximal inhibitory concentration (IC50) was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, and the cell cycle and cell proliferation were measured by flow cytometry and EdU staining, respectively. In addition, the expression of genes and proteins was quantified by quantitative reverse-transcription polymerase chain reaction (qRT–PCR) and western blotting, respectively.ResultsBoth E2F-1 and ATM expression in DDP-resistant NPC cells was much higher than that in parental cells. E2F-1 shRNA reduced ATM expression in DDP-resistant NPC cells, but ATM overexpression had no significant effect on E2F-1. ATM overexpression enhanced DDP resistance in DDP-resistant NPC cells with increased IC50 values, which was reversed by E2F-1 inhibition. Meanwhile, ATM overexpression resulted in upregulation of ABCA2 and ABCA5 in DDP-resistant NPC cells, induced elevations in the transition of the cells into S-phase, and increased cell proliferation with enhanced expression of cyclin E1, CDK2, and Ki67, which was reversed by E2F-1 shRNAs.ConclusionDownregulation of E2F-1, possibly by regulating ATM, could block the cell cycle in the G1 phase and reduce the proliferation of CNE2/DDP cells, thereby reversing the resistance of human NPC cells to DDP.

Novel sequential therapy with metformin enhances the effects of cisplatin in testicular germ cell tumours via YAP1 signalling

BackgroundTesticular germ cell tumours (TGCTs) are the most commonly diagnosed malignancy in young men. Although cisplatin has been shown to be effective to treat TGCT patients, long-term follow-up has shown that TGCT survivors who accepted cisplatin treatment suffered from a greater number of adverse reactions than patients who underwent orchiectomy alone. As metformin has shown an anticancer effect in various cancers, we investigated whether metformin could enhance the effects of cisplatin to treat TGCTs.MethodsThe anticancer effects of different treatment strategies consisting of metformin and cisplatin in TCam-2 and NTERA-2 cells were assessed in vitro and in vivo. First, we used a colony formation assay, CCK-8 and MTT assays to explore the viability of TGCT cells. Flow cytometry was used to assess the cell cycle and apoptosis of TGCTs. Then, Western blotting was used to detect the protein expression of TGCTs cells after different treatments. In addition, a xenograft model was used to investigate the effects of the different treatments on the proliferation of TGCT cells. Immunohistochemistry assays were performed to analyse the expression of related proteins in the tissues from the xenograft model.ResultsMetformin inhibited the proliferation of TCam-2 and NTERA-2 cells by arresting them in G1 phase, while metformin did not induce apoptosis in TGCT cells. Compared with cisplatin monotherapy, the CCK-8, MTT assay and colony formation assay showed that sequential treatment with metformin and cisplatin produced enhanced anticancer effects. Further study showed that metformin blocked the cells in G1 phase by inducing phosphorylated YAP1 and reducing the expression of cyclin D1, CDK6, CDK4 and RB, which enhanced the chemosensitivity of cisplatin and activated the expression of cleaved caspase 3 in TGCTs.ConclusionsOur study discovers the important role of YAP1 in TGCTs and reports a new treatment strategy that employs the sequential administration of metformin and cisplatin, which can reduce the required cisplatin dose and enhance the sensitivity of TGCT cells to cisplatin. Therefore, this sequential treatment strategy may facilitate the development of basic and clinical research for anticancer therapies to treat TGCTs.

Cell-penetrating peptides containing the progesterone receptor polyproline domain inhibits EGF signaling and cell proliferation in lung cancer cells.

Non-small cell lung cancer (NSCLC) accounts for the majority (80–85%) of all lung cancers. All current available treatments have limited efficacy. The epidermal growth factor receptor (EGFR) plays a critical role in the development and progression of NSCLC, with high EGFR expression associated with increased cell proliferation and poor prognosis. Thus, interfering with EGFR signaling has been shown to effectively reduce cell proliferation and help in the treatment of NSCLC. We previously demonstrated that the progesterone receptor (PR) contains a polyproline domain (PPD) that directly interacts with Src homology 3 (SH3) domain-containing molecules and expression of PR-PPD peptides inhibits NSCLC cell proliferation. In this study, we investigated whether the introduction of PR-PPD by cell-penetrating peptides (CPPs) could inhibit EGF-induced cell proliferation in NSCLC cells. PR-PPD was attached to a cancer-specific CPP, Buforin2 (BR2), to help deliver the PR-PPD into NSCLC cells. Interestingly, addition of BR2-2xPPD peptides containing two PR-PPD repeats was more effective in inhibiting NSCLC proliferation and significantly reduced EGF-induced phosphorylation of Erk1/2. BR2-2xPPD treatment induced cell cycle arrest by inhibiting the expression of cyclin D1 and CDK2 genes in EGFR-wild type A549 cells. Furthermore, the combination treatment of EGFR-tyrosine kinase inhibitors (TKIs), including Gefitinib or Erlotinib, with BR2-2xPPD peptides further suppressed the growth of NSCLC PC9 cells harboring EGFR mutations as compared to EGFR-TKIs treatment alone. Importantly, BR2-2xPPD peptides mediated growth inhibition in acquired Gefitinib- and Erlotinib- resistant lung adenocarcinoma cells. Our data suggests that PR-PPD is the minimal protein domain sufficient to inhibit NSCLC cell growth and has the potential to be developed as a novel NSCLC therapeutic agent.

ZNF703 promotes triple-negative breast cancer cells through cell-cycle signaling and associated with poor prognosis

BackgroundThe oncogenic drivers of triple-negative breast cancer (TNBC), which is characterized by worst prognosis compared with other subtypes, are poorly understood. Although next-generation sequencing technology has facilitated identifying potential targets, few of the findings have been translated into daily clinical practice. The present study is aimed to explore ZNF703 (Zinc finger 703) function and its underlying mechanism in TNBC.MethodsZNF703 expressions in tissue microarray were retrospectively examined by immunohistochemistry. The cell proliferation by SRB assay and colony formation assay, as well as cell cycle distribution by flow cytometry were assessed. The protein levels associated with possible underlying molecular mechanisms were evaluated by western blotting. Kaplan-Meier analysis was used to plot survival analysis.ResultsOur data suggest that ZNF703 expressed in 34.2% of triple-negative human breast tumors by immunohistochemistry. In vitro, ZNF703 knockdown had potent inhibitory effects on TNBC cell proliferation and cell cycle, with cyclin D1, CDK4, CDK6, and E2F1 downregulated, while Rb1 upregulated. Moreover, Kaplan-Meier analysis showed that high mRNA expression of ZNF703 was correlated to worse overall survival (HR for high expression was 3.04; 95% CI, 1.22 to 7.57, P = 0.017).ConclusionsTaken together, the results identified that targeting ZNF703 contributed to the anti-proliferative effects in TNBC cells, due to induced G1-phase arrest. This study is the first to identify ZNF703 as a potentially important protein that is involved in TNBC progression.

Effects of Ruxolitinib and Calcitriol Combination Treatment on Various Molecular Subtypes of Breast Cancer.

The anticancer effects of ruxolitinib and calcitriol against breast cancer were reported previously. However, the effect of ruxolitinib and calcitriol combination treatment on various molecular subtypes of breast cancer remains unexplored. In this study, we used MCF-7, SKBR3, and MDA-MB-468 cells to investigate the effect of ruxolitinib and calcitriol combination treatment on cell proliferation, apoptosis, cell cycle, and cell signaling markers, in vitro and in vivo. Our results revealed the synergistic anticancer effect of ruxolitinib and calcitriol combination treatment in SKBR3 and MDA-MB-468 cells, but not in MCF-7 cells in vitro, via cell proliferation inhibition, apoptosis induction, cell cycle arrest, and the alteration of cell signaling protein expression, including cell cycle-related (cyclin D1, CDK1, CDK4, p21, and p27), apoptosis-related (c-caspase and c-PARP), and cell proliferation-related (c-Myc, p-p53, and p-JAK2) proteins. Furthermore, in the MDA-MB-468 xenograft mouse model, we demonstrated the synergistic antitumor effect of ruxolitinib and calcitriol combination treatment, including the alteration of c-PARP, cyclin D1, and c-Myc expression, without significant drug toxicity. The combination exhibited a synergistic effect in HER2-enriched and triple-negative breast cancer subtypes. In conclusion, our results suggest different effects of the combination treatment of ruxolitinib and calcitriol depending on the molecular subtype of breast cancer.