Oridonin-induced mitochondria-dependent apoptosis in esophageal cancer cells by inhibiting PI3K/AKT/mTOR and Ras/Raf pathways

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported for its antitumor activity on several cancers. However, its effect on human esophageal cancer remains unclear. In this study, we demonstrated that oridonin could inhibit the growth of human esophageal cancer cells both in vitro and in vivo. Oridonin not only suppressed the proliferation, but also induced cell cycle arrest and mitochondrial-mediated apoptosis in KYSE-30, KYSE-150, and EC9706 cells with dose-dependent manner. Further mechanism studies revealed that oridonin led cell cycle arrest in esophageal cancer cells via downregulating cell cycle-related proteins, such as cyclin B1 and CDK2, while upregulating p53 and p21. Oridonin also increased proapoptotic protein Bax and reduced antiapoptotic protein Bcl-2, as well as the increased expression of cleaved caspase-3, -8, and -9. In addition, oridonin treatment could significantly inhibit the PI3K/Akt/mTOR and Ras/Raf signaling pathway. In vivo results further demonstrated that oridonin treatment markedly inhibited tumor growth in the esophageal cancer xenograft mice model. Taken together, these results suggest that oridonin may be a potential anticancer agent for the treatment of esophageal cancer.     

Oridonin induces G2/M arrest and apoptosis via activating ERK-p53 apoptotic pathway and inhibiting PTK-Ras-Raf-JNK survival pathway in murine fibrosarcoma L929 cells

Oridonin was reported to induce L929 cell apoptosis via ROS-mediated mitochondrial and ERK pathways; however, the precise mechanisms by which oridonin induces cell death remain unclear. Herein, we found that oridonin treatment induced an increase in G₂/M phase cell percentage. And, G₂/M phase arrest was associated with down-regulation of cell cycle related cdc2, cdc25c and cyclinB levels, as well as up-regulation of p21 and p-cdc2 levels. In addition, we discovered that interruption of p53 activation decreased oridonin-induced apoptosis, and blocking ERK by specific inhibitors or siRNA suppressed oridonin-induced p53 activation. Moreover, inhibition of PTK, protein kinase C, Ras, Raf or JNK activation increased oridonin-induced apoptosis. Also, the level of Ras, Raf or JNK was down-regulated by oridonin, and the inhibition of PTK, Ras, Raf activation decreased p-JNK level. In conclusion, oridonin induces L929 cell G₂/M arrest and apoptosis, which is regulated by promoting ERK-p53 apoptotic pathway and suppressing PTK-mediated survival pathway.     

Combined activity of oridonin and wogonin in advanced-stage ovarian cancer cells

The initial response rates of advanced-stage epithelial ovarian cancer to the chemotherapeutic agents carboplatin and paclitaxel are high. However, once drug resistance develops, further chemotherapy is less effective. The objective of this study is to investigate the anti-proliferative activity of the phyto-active chemicals (PACs) oridonin and wogonin in chemo-resistant epithelial ovarian cancer cells. Primary cell cultures from the ascitic fluid of three patients at diagnosis, two patients chemo-resistant to carboplatin and paclitaxel, and one patient treated with letrozole for breast cancer were studied and compared to the ovarian cancer cell lines A2780 and PTX10, by cell viability assay (MTS). Effects on cell cycle modulation and apoptosis were examined by flow cytometry and Western blot analysis (WB). WB was further conducted to investigate protein expressions altered by PACs. The results show that IC₅₀ of the primary cultures ranged from 0.6 to 5.4 μg/ml for oridonin and 0.3–12.7 μg/ml for wogonin. The paclitaxel-resistant cell line PTX10 was more sensitive to each of the PACs than the chemo-sensitive cell line A2780. Of particular interest is that in combination, the two PACs were synergistic in their cytotoxicity to five of six of the primary cultures and to both the cell lines (combination indices of 0.39–0.95). The inhibition is attributable to apoptosis and cell cycle modulation induced by the PACs as demonstrated in A2780 and PTX10. Up-regulation of the functional p53 protein in A2780 and down-regulation of Akt protein in PTX10 have in part contributed to the apoptosis. These findings suggest that oridonin and wogonin may have activity in ovarian cancer following its development of resistance to carboplatin and paclitaxel.     

ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small-molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a genome-wide short hairpin RNA screen for genes that are lethal in combination with p53 activation by Nutlin-3, which showed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors also enable Nutlin-3 to kill tumor spheroids. These results identify new pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies.     

Oridonin: targeting programmed cell death pathways as an anti‐tumour agent

Oridonin, an active diterpenoid isolated from traditional Chinese herbal medicine, has drawn rising attention for its remarkable apoptosis‐ and autophagy‐inducing activity and relevant molecular mechanisms in cancer therapy. Apoptosis is a well known type of cell death, whereas autophagy can play either pro‐survival or pro‐death roles in cancer cells. Accumulating evidence has recently revealed relationships between apoptosis and autophagy induced by oridonin; however, molecular mechanisms behind them remain to be discovered. In this review, we focus on highlighting updated research on oridonin‐induced cell death signalling pathways implicated in apoptosis and autophagy, in many types of cancer. In addition, we further discuss cross‐talk between apoptosis and autophagy induced by oridonin, in cancer. Taken together, these findings open new perspectives for further exploring oridonin as a potential anti‐tumour agent targeting apoptosis and autophagy, in future anti‐cancer therapeutics.     

Oridonin suppresses gastric cancer SGC-7901 cell proliferation by targeting the TNF-alpha/androgen receptor/TGF-beta signalling pathway axis

Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF-alpha/Androgen receptor/TGF-beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC-7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF-α/Androgen receptor/TGF-β signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF-α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF-β) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC-7901 cells by regulating the TNF-α /AR /TGF-β signalling pathway axis.     

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.     

Expression of coxsackie and adenovirus receptor distinguishes transitional cancer states in therapy-induced cellular senescence

Therapy-induced cellular senescence describes the phenomenon of cell cycle arrest that can be invoked in cancer cells in response to chemotherapy. Sustained proliferative arrest is often overcome as a contingent of senescent tumor cells can bypass this cell cycle restriction. The mechanism regulating cell cycle re-entry of senescent cancer cells remains poorly understood. This is the first report of the isolation and characterization of two distinct transitional states in chemotherapy-induced senescent cells that share indistinguishable morphological senescence phenotypes and are functionally classified by their ability to escape cell cycle arrest. It has been observed that cell surface expression of coxsackie and adenovirus receptor (CAR) is downregulated in cancer cells treated with chemotherapy. We show the novel use of surface CAR expression and adenoviral transduction to differentiate senescent states and also show in vivo evidence of CAR downregulation in colorectal cancer patients treated with neoadjuvant chemoradiation. This study suggests that CAR is a candidate biomarker for senescence response to antitumor therapy, and CAR expression can be used to distinguish transitional states in early senescence to study fundamental regulatory events in therapy-induced senescence.     

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.     

Oridonin inhibits the migration and epithelial‐to‐mesenchymal transition of small cell lung cancer cells by suppressing FAK‐ERK1/2 signalling pathway

Small cell lung cancer (SCLC) is a severe malignant with high morbidity; however, few effective and secure therapeutic strategy is used in current clinical practice. Oridonin is a small molecule from the traditional Chinese herb Rabdosia rubescens. This study mainly aimed to investigate the role of oridonin on inhibiting the process of H1688, a kind of small cell lung cancer cells from human. Oridonin could suppress H1688 cell proliferation and induce their apoptosis in a high dosage treatment (20 μmol/L). Meanwhile, cell migration was suppressed by oridonin (5 and 10 μmol/L) that did not affect cell proliferation and apoptosis. The expression level of E‐cadherin was significantly increased, and the expression of vimentin, snail and slug was reduced after administration of oridonin. These expression changes were associated with the suppressed integrin β1, phosphorylation of focal adhesion kinase (FAK) and ERK1/2. In addition, oridonin (5 and 10 mg/kg) inhibited tumour growth in a nude mouse model; however, HE staining revealed a certain degree of cytotoxicity in hepatic tissue after treatment oridonin (10 mg/kg). Furthermore, the concentration of alanine aminotransferase (ALP) was significantly increased and lactate dehydrogenase (LDH) was reduced after oridonin treatment (10 mg/kg). Immunohistochemical analysis further revealed that oridonin increased E‐cadherin expression and reduced vimentin and phospho‐FAK levels in vivo. These findings indicated that oridonin can inhibit the migration and epithelial‐to‐mesenchymal transition (EMT) of SCLC cells by suppressing the FAK‐ERK1/2 signalling pathway. Thus, oridonin may be a new drug candidate to offer an effect of anti‐SCLC with relative safety.     

Mechanism of T-oligo-induced cell cycle arrest in Mia-PaCa pancreatic cancer cells

DNA oligonucleotides with sequence homology to human telomeric DNA (T-oligo) induce cell cycle arrest, followed by apoptosis, senescence, or autophagy in a human cancer cell type-specific manner. T-oligo has potential as a new therapeutic strategy in oncology because of its ability to target certain types of tumor cells while sparing normal ones. In the present study, we demonstrate the T-oligo-induced S-phase cell cycle arrest in four pancreatic cancer cell lines. To further contribute to the mechanistic understanding of T-oligo, we also identify cyclin dependent kinase 2 (cdk2) as a functional mediator in the T-oligo-induced cell cycle arrest of pancreatic cancer cells. Ectopic expression of a constitutively active cdk2 mutant abrogates T-oligo-induced cell cycle arrest in these tumor cells while knockdown of cdk2 expression alone recapitulates the T-oligo effect. Finally, we demonstrate the dispensability of T-oligo-induced ATM/ATR-mediated DNA damage response-signaling pathways, which have long been considered functional in the T-oligo signaling mechanism.     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Oridonin suppress cell migration via regulation of nonmuscle myosin IIA

Oridonin, which is isolated from Chinese herb Rabdosia rubescens (Hemsl.) Hara, has been implicated in regulation of tumor cell migration and invasion. In this study, treatment with oridonin enhanced the phosphorylation of myosin regulatory light chain (T18/S19) that regulates the ATPase activity of myosin IIA. Meanwhile, stress fibers were significantly more prominent after oridonin incubation, which impaired cell migration in transwell migration assays. All of these effects may be caused by the decreased interaction between myosin IIA and myosin phosphatase complex, but not kinases. Our data provide clear evidence of this novel pharmacological function for oridonin in treating cancer cell migration.     

Garlic Compound, Diallyl Disulfide Induces Cell Cycle Arrest in Prostate Cancer Cell Line PC-3

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. Previous studies reported that aged garlic extract suppresses cancer growth. In the present study, diallyl disulfide [DADS], oil soluble organosulfur compound of garlic, was studied for its antiproliferative and induction of cell cycle arrest on prostate cancer cells in vitro. The suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed and confirmed by propidium iodide staining in flowcytometric analysis and western blotting analysis of major cell cycle regulator proteins. The results showed that DADS inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 25 μM and 40 μM concentrations, DADS induced cell cycle arrest at G2/M transition in PC-3 cells. Western blotting analysis of cyclin A, B₁ and cyclin dependent kinase 1 [CDK1] revealed that DADS inhibited the cell cycle by downregulating CDK1 expression. It is concluded that DADS, inhibits proliferation of prostate cancer cells through cell cycle arrest. Dose dependent effect of DADS on PC-3 cell line was observed in the present study.     

Therapeutic targeting of cancer cell cycle using proteasome inhibitors

Proteasomes are multicatalytic protease complexes in the cell, involved in the non-lysosomal recycling of intra-cellular proteins. Proteasomes play a critical role in regulation of cell division in both normal as well as cancer cells. In cancer cells this homeostatic function is deregulated leading to the hyperactivation of the proteasomes. Proteasome inhibitors (PIs) are a class of compounds, which either reversibly or irreversibly block the activity of proteasomes and induce cancer cell death. Interference of PIs with the ubiquitin proteasome pathway (UPP) involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI induced cell cycle arrest in a variety of cancer cells. Although many PIs target the proteasomes, not all of them are effective in cancer therapy. Some cancers develop resistance against proteasome inhibition by possibly activating compensatory signaling pathways. However, the details of the activation of these pathways and their contribution to resistance to PI therapy remain obscure. Delineation of these pathways may help in checking resistance against PIs and deducing effective combinational approaches for improved treatment strategies. This review will discuss some of the signaling pathways related to proteasome inhibition and cell division that may help explain the basis of resistance of some cancers to proteasome inhibitors and underline the need for usage of PIs in combination with traditional chemotherapy.     

Synthesis and biological evaluation of novel 1-O- and 14-O-derivatives of oridonin as potential anticancer drug candidates

Novel 1-O- and 14-O-derivatives of oridonin were synthesized and biologically evaluated. All of the derivatives exhibited stronger cytotoxicity against six cancer cell lines (BGC-7901, SW-480, HL-60, BEL-7402, A549, and B16) than oridonin in vitro, and some of them were more potent than oridonin and cyclophosphamide in vivo. Compounds Ib and IIg were the most potent with the IC(50) values of 0.84 microM for Ib in HL-60 cell and 1.00 microM for IIg in BEL-7402 cell.