Cyclin CDK4 Research Articles

Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation

ObjectiveProtein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. MethodsGC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. ResultsThe expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. ConclusionPDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.

Novel cyanochalcones as potential anticancer agents: apoptosis, cell cycle arrest, DNA binding, and molecular docking studies

In the light of anticancer drug discovery and development, a new series of cyanochalcones incorporating indole moiety (5a-g) were efficiently synthesized and characterized by different spectral analysis. MTT assay was used to evaluate the antiproliferative activity of the synthesized compounds towards different cancer cells (Hela, MDA-MB-231, A375, and A549) in parallel with normal cells (HSF). Trimethoxy and diethoxy-containing derivatives (5d and 5e) displayed the most selective cytotoxic activities against cervical Hela cells with IC50 values of 8.29 and 11.82 µM, respectively, with great safety pattern toward normal HSF cells (Selectivity index: 21.3 and 13.9, respectively). Therefore, 5d and 5e were chosen to study their effects on apoptosis, cell cycle arrest, and migration of Hela cells using flow cytometric analysis and wound healing assay. They induced apoptosis and cell cycle arrest at the S phase and impaired migration of HeLa cells. Regarding their effects on the expression profile of crucial genes related to the potential anticancer activities, 5d and 5e remarkably upregulated caspase 3 and Beclin1 and downregulated cyclin A1, CDK2, CDH2, MMP9, and HIF1A using qRT-PCR and ELISA techniques. UV–Vis spectral measurement demonstrated the ability of 5d and 5e to bind CT-DNA efficiently with Kb values of 3.7 × 105 and 1 × 105 M−1, respectively. Moreover, in silico molecular docking was performed to assess the binding affinities of the compounds toward the active sites of Bcl2, CDK2, and DNA. Therefore, cyanochalcones 5d and 5e might be promising anticancer agents and could offer a scientific basis for intensive research into cancer chemotherapy. Communicated by Ramaswamy H. Sarma

Green-Synthesized Silver and Selenium Nanoparticles Using Berberine: A Comparative Assessment of In Vitro Anticancer Potential on Human Hepatocellular Carcinoma Cell Line (HepG2).

A well-known natural ingredient found in several medicinal plants, berberine (Ber), has been shown to have anticancer properties against a range of malignancies. The limited solubility and bioavailability of berberine can be addressed using Ber-loaded nanoparticles. In this study, we compared the in vitro cytotoxic effects of both Ber-loaded silver nanoparticles (Ber-AgNPs) and Ber-loaded selenium nanoparticles (Ber-SeNPs) in the human liver cancer cell line (HepG2) and mouse normal liver cells (BNL). The IC50 values in HepG2 for berberine, Ber-AgNPs, Ber-SeNPs, and cisplatin were 26.69, 1.16, 0.04, and 0.33 µg/mL, respectively. Our results show that Ber and its Ag and Se nanoparticles exerted a good antitumor effect against HepG2 cells by inducing apoptosis via upregulating p53, Bax, cytosolic cytochrome C levels, and caspase-3 activity, and the down-regulation of Bcl-2 levels. Similarly, incubation with Ber and both Ber-NPs (Ag and Se) led to a significant dose-dependent elevation in inflammatory markers' (TNF-α, NF-κB, and COX-2) levels compared to the control group. In addition, it led to the arrest of the G1 cell cycle by depleting the expression of cyclin D1 and CDK-2 mRNA. Furthermore, Ber and both Ber-NPs (Ag and Se) caused a significant dose-dependent increase in LDH activity in HepG2 cells. Furthermore, our findings offer evidence that Ber and its nanoparticles intensified oxidative stress in HepG2 cells. Furthermore, the migration rate of cells subjected to berberine and its nanoforms was notably decreased compared to that of control cells. It can be inferred that Ber nanoparticles exhibited superior anticancer efficacy against HepG2 compared to unprocessed Ber, perhaps due to their improved solubility and bioavailability. Furthermore, Ber-SeNPs exhibited greater efficacy than Ber-AgNPs, possibly as a result of the inherent anticancer characteristics of selenium.

Exploring the Anticancer Potential of Traditional Thai Medicinal Plants: A Focus on Dracaena loureiri and Its Effects on Non-Small-Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC) is renowned for its aggressive and highly metastatic nature. In recent years, there has been a surge in interest regarding the therapeutic potential of traditional medicinal plants. Dracaena loureirin (D. loureirin), Ficus racemosa Linn. (F. racemosa), and Harrisonia perforata (Blanco) Merr. (H. perforata) are prominent traditional medicinal herbs in Thailand, recognized for their diverse biological activities, including antipyretic and anti-inflammatory effects. However, their prospective anti-cancer properties against NSCLC remain largely unexplored. This study aimed to evaluate the anti-cancer attributes of ethanolic extracts obtained from D. loureiri (DLEE), F. racemosa (FREE), and H. perforata (HPEE) against the A549 lung adenocarcinoma cell lines. Sulforhodamine B (SRB) assay results revealed that only DLEE exhibited cytotoxic effects on A549 cells, whereas FREE and HPEE showed no such cytotoxicity. To elucidate the anti-cancer mechanisms of DLEE, cell cycle and apoptosis assays were performed. The findings demonstrated that DLEE inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase in A549 cells through the downregulation of key cell cycle regulator proteins, including cyclin D1, CDK-2, and CDK-4. Furthermore, DLEE treatment facilitated apoptosis in A549 cells by suppressing anti-apoptotic proteins (Bcl-2, Bcl-xl, and survivin) and enhancing apoptotic proteins (cleaved-caspase-3 and cleaved-PARP-1). In summary, our study provides novel insights into the significant anti-cancer properties of DLEE against A549 cells. This work represents the first report suggesting that DLEE has the capability to impede the growth of A549 lung adenocarcinoma cells through the induction of apoptosis.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

Morroniside induces cardiomyocyte cell cycle activity and promotes cardiac repair after myocardial infarction in adult rats.

Introduction: Acute myocardial infarction (AMI) is characterized by the loss of cardiomyocytes, which impairs cardiac function and eventually leads to heart failure. The induction of cardiomyocyte cell cycle activity provides a new treatment strategy for the repair of heart damage. Our previous study demonstrated that morroniside exerts cardioprotective effects. This study investigated the effects and underlying mechanisms of action of morroniside on cardiomyocyte cell cycle activity and cardiac repair following AMI. Methods: Neonatal rat cardiomyocytes (NRCMs) were isolated and exposed to oxygen-glucose deprivation (OGD) in vitro. A rat model of AMI was established by ligation of the left anterior descending coronary artery (LAD) in vivo. Immunofluorescence staining was performed to detect newly generated cardiomyocytes. Western blotting was performed to assess the expression of cell cycle-related proteins. Electrocardiography (ECG) was used to examine pathological Q waves. Masson's trichrome and wheat germ agglutinin (WGA) staining assessed myocardial fibrosis and hypertrophy. Results: The results showed that morroniside induced cardiomyocyte cell cycle activity and increased the levels of cell cycle proteins, including cyclin D1, CDK4, cyclin A2, and cyclin B1, both in vitro and in vivo. Moreover, morroniside reduced myocardial fibrosis and remodeling. Discussion: In conclusion, our study demonstrated that morroniside stimulates cardiomyocyte cell cycle activity and cardiac repair in adult rats, and that these effects may be related to the upregulation of cell cycle proteins.

SLL-1A-16 suppresses proliferation and induces autophagy in non-small-cell lung cancer cells via the AKT/mTOR signaling pathway.

Non-small-cell lung cancer (NSCLC), which accounts for approximately eighty-five percent of lung cancer diagnoses worldwide, is a malignancy with high incidence and mortality rates. Among the various antitumor compounds, organic selenium-containing compounds have emerged as a promising class of therapeutic agents for cancer treatment. In the present study, SLL-1A-16, a new organoselenium small molecule, was discovered to exhibit antiproliferative activity against NSCLC both in vitro and in vivo. Treatment with SLL-1A-16 significantly inhibited NSCLC cell proliferation and induced apoptosis and autophagy. Mechanistically, SLL-1A-16 inhibited cell proliferation through G1-S phase arrest by reducing cyclin D1 and CDK4 expression. Additionally, SLL-1A-16 significantly induced apoptosis by upregulating cleaved caspase 3 and Bax expression, while downregulating Bcl-2 levels. Our study also demonstrated that SLL-1A-16 induced autophagy in NSCLC cells by inhibiting the Akt/mTOR pathway. Overall, our findings suggest that SLL-1A-16 could induce cell cycle arrest, apoptosis and autophagy in NSCLC cells by inhibiting the Akt/mTOR signaling pathways, providing a theoretical basis for the potential clinical application of SLL-1A-16 as a chemotherapeutic agent in NSCLC treatment.

Antiproliferative Activity of Gibbosic Acid H through Induction of G0/G1 Cell Cycle Arrest and Apoptosis in Human Lung Cancer Cells.

Lung cancer is one of the most common causative cancers worldwide. Particularly, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. NSCLC is a serious form of lung cancer that requires prompt diagnosis, and the 5-year survival rate for patients with this disease is only 24%. Gibbosic acid H (GaH), a natural lanostanoid obtained from the Ganoderma species (Ganodermataceae), has antiproliferative activities against colon and lung cancer cells. The aim of the present study was to evaluate the antiproliferative activity of GaH in NSCLC cells and to elucidate the underlying molecular mechanisms. GaH was found to induce G0/G1 cell cycle arrest and autophagy by activating adenosine monophosphate-activated protein kinase in A549 and H1299 cells. The induction of this cell cycle arrest was associated with the downregulation of cyclin E1 and CDK2. Additionally, the induction of autophagy by GaH was correlated with the upregulation of LC3B, beclin-1, and p53 expression. GaH also induced apoptosis by upregulating cleaved caspase-3 and Bax in the lung cancer cells. These findings suggest that GaH has a potential in the growth inhibition of human lung cancer cells.

NEK2 overexpression aggravates IL-22-induced keratinocyte proliferation and cytokine level increases and IMQ-induced psoriasis-like dermatitis.

Psoriasis is a common inflammatory skin disease characterized by the excessive proliferation and abnormal differentiation of keratinocytes. Protein kinases could act on intracellular signaling pathways associated with cell proliferation. Identifying more hub protein kinases affecting cellular and molecular processes in psoriasis, and exploring the dynamic effects of baicalin and NEK2 on the IL-22-induced cellular inflammation and IMQ-induced psoriasis-like mice. In this study, differentially expressed protein kinases playing a hub role in psoriasis initiation and development were identified using integrative bioinformatics analyses, and NEK2 has been chosen. NEK2 was significantly up-regulated in psoriatic samples according to online datasets and experimental analyses. In IL-22-induced cellular inflammation model in HaCaT cells, NEK2 overexpression promoted, whereas NEK2 knockdown partially abolished IL-22-induced alterations in cell viability, DNA synthesis, cytokine levels, as well as STAT3 phosphorylation and p-RB, cyclin D1, CDK4, and CDK6 protein contents. Baicalin treatment partially suppressed IL-22-induced HaCaT cell viability, DNA synthesis, and increases in cytokine levels, whereas NEK2 overexpression significantly abolished Baicalin-induced protection against cellular inflammation. In IMQ-induced psoriasis-like skin inflammation model in mice, baicalin markedly ameliorated IMQ-induced psoriasis-like symptoms and local skin inflammation, whereas NEK2 overexpression partially eliminated the therapeutic effects of baicalin. NEK2, up-regulated in psoriatic lesion skin, could aggravate IMQ-induced psoriasis-like dermatitis and attenuate the therapeutic efficiency of baicalin through promoting keratinocyte proliferation and cytokine levels. The STAT3 signaling might be involved.

Harnessing Btki Therapy By CDK4/6i Control of T Effector Memory Cells and T Cell Surveillance in Mantle Cell Lymphoma

Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL, providing a rationale for targeting the cell cycle in MCL therapy. We have demonstrated in preclinical studies that inhibition of CDK4/6 not only induces early G1 cell cycle arrest, but also reprograms MCL cells for cytotoxic killing. On this basis, we investigated if inhibition of cyclin D1/CDK4 with palbociclib (CDK4/6 inhibitor) could reprogram recurrent MCL to deepen and prolong the ibrutinib (BTK inhibitor) response in a phase I clinical trial, and the resistance mechanism by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Palbociclib was administered to MCL patients on days 1-21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before and during therapy, and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node (53 samples) was performed using a unique in house MCL cell-specific library. PBMC from normal donors (n=4) and untreated MCL patients (n=4) served as controls. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of MCL cells isolated from the same specimens, as well as flowcytometry in conjunction with IHC. Total numbers of MCL and immune cells in each specimen were deduced by integrating complete blood counts (CBC) with differential with scRNA-seq or flowcytometry. CDK4/6 Inhibition appears to deepen and prolong the BTKi response, with a CR rate of 42% and 5 patients (2 CR and 3 PR) remained on therapy for ~ 9 years. Longitudinal scRNA-seq (210,000 cells) revealed that MCL cells comprise 4 major transcriptomically distinct clusters. Cluster 1 (C1) resembles quiescent normal B cells; C2 mimics hyper-activated B cells that are enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative. RNA velocity analysis further indicates that having progressed to late G1 after evading CDK4/6 inhibition, MCL cells in C2 are in transit to C4. By contrast, C3 is a terminal node of MCL cells arrested in late G1 with elevated BCL2 expression Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 MCL cells, or C2 MCL cells that fuel the proliferating C4 MCL cells. Resistance was also associated with a profound reduction in MHC I and MHC II expression on MCL cells in some patients. Moreover, concurrent with disease progression, CD8+ T and CD+T cells were rapidly depleted by different mechanisms ( Figure 1). CD8+ T cells were proliferating and eliminated by exhaustion, as evidenced by the marked expression of LAG3 TIGIT and TIM3, and a failure to maintain CD8+ T effector memory cells. By contrast, CD4+ T cells were not proliferating and appeared die from glucose starvation. Collectively, these data suggest that T cell maintenance and surveillance is pivotal in sustaining a durable clinical response to combined inhibition of CDK4/6 and BTK. Guided by these discoveries, we have now restored BTKi sensitivity ex vivo in MCL cells from resistant patientsby targeting patient-specific expansion of C2 or C3 in vivo. In one resistant patient, this led to the maintenance of CD4+ and CD8+ T effector memory cells and a CR in response to venetoclax plus BTKi for nearly 3 years and continuing. In summary, by integrated longitudinal scRNA-seq analysis of a hypothesis-driven therapy, we have provided the first evidence that 1) MCL cells comprise 4 major transcriptomically distinct clusters; 2) resistance to CDK4/6i is associated with expansion of C2 that fuels the proliferating C4 MCL cells, or the long-lived C3 MCL cells with elevated BCL2 expression; 3) CDK4/6i harnesses BTKi response by promoting the maintenance of effector memory T cells; 4) combined inhibition of BCL2 and BTK overrides resistance to CDK4/6i and BTKi, leading to a durable clinical response; and 5) T cell surveillance is pivotal for a durable response to CDK4/6, BTK or BCL2 inhibition; implicating genome-guided combination therapy to overcome therapy resistance in MCL.

CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas.

CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.

Bruceine A: Suppressing metastasis via MEK/ERK pathway and invoking mitochondrial apoptosis in triple-negative breast cancer

Triple-negative breast cancer (TNBC), as the most aggressive subtype of breast cancer, presents a scarcity of miraculous drugs in suppressing its proliferation and metastasis. Bruceine A (BA) is a functional group-rich quassin compound with extensive and distinctive pharmacological activities. Within the present study, we investigated the capabilities of BA in suppressing TNBC proliferation and metastasis as well as its potential mechanisms. The results displayed that BA dramatically repressed the proliferation of MDA-MB-231 and 4T1 cells with corresponding IC50 values of 78.4 nM and 524.6 nM, respectively. Concurrently, BA arrested cells in G1 phase by downregulating cycle-related proteins Cyclin D1 and CDK4. Furthermore, BA distinctly induced mitochondrial dysfunction as manifested by diminished mitochondrial membrane potential, elevated reactive oxygen species generation, minimized ATP production, and Caspase-dependent activation of the mitochondrial apoptosis pathway. Additionally, BA restrained the invasion and metastasis of TNBC cells by repressing MMP9 and MMP2 expression. Intriguingly, after pretreatment with MEK activator C16-PAF, the inhibitory effect of BA on MEK/ERK pathway was notably diminished, while the proliferation suppression and metastasis repression exerted by BA were all strikingly curtailed. Molecular docking illustrated that BA potently combined with residues on the MEK1 protein with the presence of diverse intermolecular interactions. Ultimately, BA effectively suppressed tumor growth in the 4T1 xenograft tumor model with no detectable visceral toxicity in the high-dose group and, astonishingly, repressed tumor metastasis in the 4T1-luc lung metastasis model. Collectively, our study demonstrates that BA is a promising chemotherapeutic agent for treating TNBC and suppressing lung metastasis.

Expression of Cyclin E1 in hepatic stellate cells is critical for the induction and progression of liver fibrosis and hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) is one of the most severe malignancies with increasing incidence and limited treatment options. Typically, HCC develops during a multistep process involving chronic liver inflammation and liver fibrosis. The latter is characterized by the accumulation of extracellular matrix produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs in an ordered sequence that is highly regulated by cyclins and associated cyclin-dependent kinases (CDKs) such as the Cyclin E1 (CCNE1)/CDK2 kinase complex. In the present study, we examined the role of Cyclin E1 (Ccne1) and Cdk2 genes in HSCs for liver fibrogenesis and hepatocarcinogenesis. To this end, we generated conditional knockout mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1∆HSC or Cdk2∆HSC). Ccne1∆HSC mice showed significantly reduced liver fibrosis formation and attenuated HSC activation in the carbon tetrachloride (CCl4) model. In a combined model of fibrosis-driven hepatocarcinogenesis, Ccne1∆HSC mice revealed decreased HSC activation even after long-term observation and substantially reduced tumor load in the liver when compared to wild-type controls. Importantly, the deletion of Cdk2 in HSCs also resulted in attenuated liver fibrosis after chronic CCl4 treatment. Single-cell RNA sequencing revealed that only a small fraction of HSCs expressed Ccne1/Cdk2 at a distinct time point after CCl4 treatment. In summary, we provide evidence that Ccne1 expression in a small population of HSCs is sufficient to trigger extensive liver fibrosis and hepatocarcinogenesis in a Cdk2-dependent manner. Thus, HSC-specific targeting of Ccne1 or Cdk2 in patients with liver fibrosis and high risk for HCC development could be therapeutically beneficial.

Houttuynia cordata Thunb. Hexane fraction induces MDA-MB-231 cell apoptosis via caspases, ER stress, cell cycle arrest and attenuated Akt/ERK signaling

Houttuynia cordata Thunb. (HCT) is a perennial plant used in traditional Thai medicine for many centuries. This study aimed to investigate the antiproliferative effect of the hexane fraction, which has not been explored before. HCT ethanol extract (crude extract) was sequentially fractionated to obtain a hexane (H) fraction. GC-MS was used to determine the phytochemicals. The H fraction consisted of lipids, mainly α-linolenic acid and some terpenoids. MTT assay was used to determine the cytotoxic effects of H fraction in MCF-7, MDA-MB-231, NIH3T3 and PBMCs. The mode of cell death and cell cycle analysis were determined by flow cytometry. The mechanisms of cell death were defined by mitochondrial transmembrane potential (MTP) reduction and activation of caspase-3, -8 and -9. The expression levels of the Bcl-2 family, cell cycle-related, endoplasmic reticulum (ER) stress-associated proteins; and Akt/ERK signaling molecules were investigated by immunoblotting. The H fraction was toxic to MDA-MB-231 more than MCF-7 cells but not to NIH3T3 and PBMCs. The growth of MDA-MB-231 cells was inhibited through apoptosis. MTP was disrupted whereas caspase-3, -8 and -9 were activated. The expression of pro-apoptotic Bax and Bak was upregulated, while Bid and anti-apoptotic Bcl-xL proteins were downregulated. Cyclin D1 and CDK4 levels were downregulated. The cell cycle was arrested at G1. Moreover, GRP78 and CHOP elevation indicated ER stress-mediated pathway. The expression ratio of pAkt/Akt and pERK/ERK were reduced. Taken together, the molecular mechanisms of MDA-MB-231 cell apoptosis were via intrinsic/extrinsic pathways, cell cycle arrest, ER stress and abrogation of Akt/ERK survival pathways. According to the most current research, the H fraction may be used as an adjuvant in the BC treatment; however, before the anticancer strategy can be applied to patients, it is important to determine each active compound's effects in cell lines and in vivo when compared with a combined mixture.

Lenalidomide arrests cell cycle and modulates PD1-dependent downstream mTOR intracellular signals in melanoma cells.

Despite numerous efforts to define the best therapeutic strategies in advanced melanoma, the response of many patients remains heterogeneous and of short duration. Lenalidomide, an immunomodulating drug, has shown anti-inflammatory, antiangiogenic and anticancer properties in haematological disorders; however, few preclinical data support the rationale for using this drug in melanoma patients. In this study, we investigate lenalidomide's potential role in melanoma by focusing on the in-vitro drug's antiproliferative activity. The antiproliferative action of lenalidomide was evaluated on two melanoma cell lines by MTT assay, cell cycle and apoptosis assay. P21 protein levels were evaluated with droplet digital PCR (ddPCR) and western blot analysis while his interaction with specific cyclin-dependent kinase (CDK) was assessed by immunoprecipitation test. The biological effect and molecular mechanisms of programmed cell death-1 (PD-1) in the regulation of proliferation were evaluated using ddPCR, flow cytometry, western blot and small interfering RNA transfection. We observed that lenalidomide exerts a cytostatic effect in melanoma cell lines by inducing cell cycle arrest in the G0-G1 phase through p21 upregulation and modulation of CDK complexes. Furthermore, we found that lenalidomide has an antiproliferative action through the downregulation of melanoma-PD1 expression and consequently the alteration of intracellular signaling of mammalian target of rapamycin/S6. The present study aims to provide new insights into the role of lenalidomide in melanoma and suggesting to potentially translating these findings into a clinical setting to use immunomodulatory derivatives for blocking the pro-tumorigenic activity of the melanoma through the PD-1/PD-L1 axis.

Advance on cyclin D1 and CDK4 in cutaneous melanoma

Advance on cyclin D1 and CDK4 in cutaneous melanoma

Overexpression of long non-coding RNA GASL1 induces apoptosis and G0/G1 cell cycle arrest in human oral cancer cells

Oral cancer is one of the commonly reported malignancies of the human oral cavity and pharynx. It accounts for a significant level of cancer-based mortality across the globe. Long non-coding RNAs (lncRNAs) are emerging as important study targets in cancer therapy. The present study aimed to characterize the role of lncRNA GASL1 in regulating the growth, migration, and invasion of human oral cancer cells. The qRT-PCR showed significant (P&lt;0.05) upregulation of GASL1 in oral cancer cells. Overexpression of GASL1 led to the loss of viability of HN6 oral cancer cells by inducing apoptosis which was associated with upregulation of Bax and downregulation of Bcl-2. The apoptotic cell percentage increased from 2. 81% in control to 25.89% upon GASL1 overexpression. Cell cycle analysis showed that overexpression of GASL1 increased the G1 cells from 35.19% in control to 84.52% upon GASL1 overexpression indicative of G0/G1 cell cycle arrest. Cell cycle arrest was also accompanied by inhibition of cyclin D1 and CDK4 protein expression. Wound healing and transwell assays showed that overexpression of GASL1 significantly (P&lt;0.05) inhibited the migration and invasion of HN6 oral cancer cells. The invasion of the HN6 oral cancer cells was found to be decreased by more than 70%. Finally, the results of in vivo study revealed that GASL1 overexpression inhibits the xenografted tumor growth in vivo. Thus, the results are thus suggestive of the tumor-suppressive molecular role of GASL1 in oral cancer cells.

HARNESSING BTKI THERAPY BY CDK4/6I CONTROL OF T CELL SURVEILLANCE

Introduction: Drug resistance remains a formidable challenge in mantle cell lymphoma (MCL). Cell cycle dysregulation driven by aberrant Cyclin D1 and CDK4 expression is a hallmark for MCL. By inhibition of CDK4/6, we have developed a novel strategy that both inhibits proliferation of MCL cells and reprograms them for cytotoxic killing. In a phase I clinical trial we investigated if inhibition of cyclin D1/CDK4 with palbociclib could reprogram recurrent MCL to deepen and prolong the ibrutinib response, and the mechanism for resistance by longitudinal genomic analysis of sequential samples from individual patients in the context of the clinical response. Methods: Palbociclib was administered to recurrent MCL patients on days 1–21 of a 28-day cycle; ibrutinib was given continuously. For longitudinal genomic analysis, sequential tissue and blood specimens from 27 evaluable MCL patients before, during therapy and on progression were collected. Single cell RNA-seq (scRNA-seq) of PBMC or the monocytic fractions from bone marrow and lymph node was performed using a unique in house MCL-specific library. The data were then subject to multiplex analysis with whole transcriptome sequencing and whole exome sequencing of purified MCL cells and flowcytometry of the same samples in conjunction with IHC. Results: Palbociclib appears to deepen and prolong the ibrutinib response; the CR rate was 42% CR, and 5 patients (2 CR and 3 PR) remained on therapy for >8 years. Longitudinal scRNA-seq further revealed that MCL cells comprise 4 major transcriptomically distinct clusters (C)s. Primary resistance and progression on therapy were associated with a marked expansion of either long-live non-proliferating C3 cells or C2 cells that fuel the proliferating C4 cells. Resistance was associated with a profound reduction in MHC I and MHC II expression and disease progression was further accompanied by a rapid loss of both CD8+ and CD+ T cells. This suggests T cell surveillance plays a critical role in maintaining a durable ibrutinib response. Guided by our discoveries, we have restored ibrutinib sensitivity by target patient-specific expansion of C2 or C3 MCL cells ex vivo. In one patient, this led to restoration of CD4+ and CD8+T cells and a CR over 2.5 years. Conclusion: T cell surveillance plays a critical role in prolonging the ibrutinib response by CDK4/6 inhibition, implicating genome-guided combination therapy to overcome ibrutinib resistance in MCL. The research was funded by: PO1 grant from the National Cancer Institute MCL-RI grant from Leukemia and Lymphoma Society Keywords: combination therapies, genomics, epigenomics, and other-omics, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Effects of TCPP and TCEP exposure on human corneal epithelial cells: Oxidative damage, cell cycle arrest, and pyroptosis

Tris(2-chloroisopropyl) phosphate (TCPP) and Tris(2-chloroethyl) phosphate (TCEP) are the widely used organophosphorus flame retardants indoors and easily accessible to the eyes as the common adhesive components of dust and particle matter, however, hardly any evidence has demonstrated their corneal toxicity. In this study, the adverse effects of TCPP, TCEP, and TCPP + TCEP exposure on human corneal epithelial cells (HCECs) were investigated. The cell viability and morphology, intracellular reactive oxygen species (ROS), cell cycle, and the expressions of cell cycle and pyroptosis-related genes were assessed to explain the underlying mechanisms. Compared to individual exposure, co-exposure to TCPP20+TCEP20 showed higher cytotoxicity with a sharp decrease of >30% in viability and more serious oxidative damage by increasing ROS production to 110.92% compared to the control group. Furthermore, the cell cycle arrested at the S phase (36.20%) was observed after combined treatment, evidenced by the upregulation of cyclin D1, CDK2, CDK4, CDK6, p21, and p27. Interestingly, pyroptosis-related genes GSDMD, Caspase-1, NLRP3, IL-1β, IL-18, NLRP1, and NLRC4 expressions were promoted with cell swelling and glowing morphology. Oxidative stress and cell cycle arrest probably acted as a key role in TCPP20+TCEP20-induced cytotoxicity and pyroptosis in HCECs. Our results suggested that TCPP20+TCEP20 co-exposure induced severer corneal damage, further illustrating its significance in estimating indoor health hazards to humans.

Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant (pho81Δ) and a constitutively activated PHO pathway mutant (pho80Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in pho80Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP). Elevated phosphate in pho80Δ coincided with elevated metal ions, metal stress sensitivity, and a muted calcineurin response, all of which were ameliorated by phosphate depletion. In contrast, metal ion homeostasis was largely unaffected in the pho81Δ mutant, and Pi, polyP, ATP, and energy metabolism were reduced, even under phosphate-replete conditions. A similar decline in polyP and ATP suggests that polyP supplies phosphate for energy production even when phosphate is available. Using calcineurin reporter strains in the wild-type, pho80Δ, and pho81Δ background, we also demonstrate that phosphate deprivation stimulates calcineurin activation, most likely by increasing the bioavailability of calcium. Finally, we show that blocking, as opposed to permanently activating, the PHO pathway reduced fungal virulence in mouse infection models to a greater extent and that this is most likely attributable to depleted phosphate stores and ATP, and compromised cellular bioenergetics, irrespective of phosphate availability. IMPORTANCE Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited. In contrast to humans, fungal cells maintain phosphate homeostasis via a CDK complex, providing drug-targeting opportunities. To investigate which CDK components are the best targets for potential antifungal therapy, we used strains with a constitutively active (pho80Δ) and an activation-defective (pho81Δ) PHO pathway, to investigate the impact of dysregulated phosphate homeostasis on cellular function and virulence. Our studies suggest that inhibiting the function of Pho81, which has no human homologue, would have the most detrimental impact on fungal growth in the host due to depletion of phosphate stores and ATP, irrespective of phosphate availability in the host.