CDK4/6 inhibitor palbociclib promotes SARS-CoV-2 cell entry by down-regulating SKP2 dependent ACE2 degradation.

Coronavirus disease 2019 (COVID-19) outbreak has become a global pandemic. CDK4/6 inhibitor palbociclib was reported to be one of the top-scored repurposed drugs to treat COVID-19. As the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry, expression level of angiotensin-converting enzyme 2 (ACE2) is closely related to SARS-CoV-2 infection. In this study, we demonstrated that palbociclib and other methods could arrest cells in G0/G1 phase and up-regulate ACE2 mRNA and protein levels without altering its subcellular localization. Palbociclib inhibited ubiquitin-proteasome and lysosomal degradation of ACE2 through down-regulating S-phase kinase-associated protein 2 (SKP2). In addition, increased ACE2 expression induced by palbociclib and other cell cycle arresting compounds facilitated pseudotyped SARS-CoV-2 infection. This study suggested that ACE2 expression was down-regulated in proliferating cells. Cell cycle arresting compounds could increase ACE2 expression and facilitate SARS-CoV-2 cell entry, which may not be suitable therapeutic agents for the treatment of SARS-CoV-2 infection.

Inhibition of fatty acid synthase (FASN) affects the proliferation and apoptosis of HepG2 hepatoma carcinoma cells via the ß-catenin/C-myc signaling pathway.

INTRODUCTION AND OBJECTIVES: Research in the last few years has proven that inhibition of fatty acid synthase (FASN) suppresses the migration and invasion of hepatoma carcinoma cells. This study aims to explore the effect of fatty acid synthase knockdown on the apoptosis and proliferation of HepG2 cells. MATERIALS AND METHODS: The human liver cancer cell line HepG2 was cultured and then transfected with FASN-specific siRNA and negative control RNAi. After 48h, cells and protein lysates were used for western blotting, CCK-8 (cell counting kit-8) assays, flow cytometry and other tests. To assess cell apoptosis, Bax, Bcl-2 and caspase-3 were detected; to assess proliferation, CDK4 (cyclin-dependent kinases 4) and P21 were detected; and to determine the signaling pathway involved, ß-catenin and C-myc were also detected. RESULTS: Inhibition of FASN in HepG2 cells can decrease proliferation and promote apoptosis. Flow cytometry and CCK-8 assays demonstrated that the apoptosis rate of FASN-specific siRNA-transfected cells was significantly increased compared to that of the control cells (p<0.01). In addition, the cell cycle analysis revealed that FASN-specific siRNA-transfected cells induced G1 phase arrest (p<0.05), but an increasing trend in G2 (p<0.05). Compared with expression in negative RNAi-transfected cells, the expression of Bcl-2 and CDK-4 was reduced and the expression of Bax, caspase-3 and P21 was increased in FASN-specific siRNA-transfected cells (p<0.05). Regarding the signaling pathway, the expression of ß-catenin and C-myc was significantly reduced when compared to that in negative control cells (p<0.05). CONCLUSIONS: Inhibition of FASN suppressed the cell survival of HepG2 cells by inhibiting the ß-catenin/C-myc pathway. This result suggests the potential treatment value of FASN for hepatoma carcinoma (HCC).

Curcumin Nanoemulsions Stabilized with Modified Phosphatidylcholine on Skin Carcinogenesis Protocol.

BACKGROUND: Cancer is one of the main causes of death by disease; several alternative treatments have been developed to counteract this condition. Curcumin (diferuloylmethane), extracted from the rhizome of Curcuma longa, has antioxidant, anti-inflammatory, and anti-cancer properties; however, it has low water solubility and poor intestinal absorption. Carrier systems, such as nanoemulsions, can increase the bioavailability of lipophilic bioactive compounds. OBJECTIVE: To evaluate the effect of curcumin nanoemulsions prepared with lecithin modified with medium-chain fatty acids as an emulsifier, on the expression of the Cdk4, Ccne2, Casp8 and Cldn4 genes involved in the carcinogenesis process in K14E6 transgenic mice. METHODS: The emulsifier was prepared by interesterification of medium-chain fatty acids, pure lecithin, and immobilized phospholipase-1 on Duolite A568. An Ultraturrax homogenizer and a Branson Ultrasonic processor were used for the preparation of nano-emulsions, and a Zetasizer evaluated the particle size. qRT-PCR analysis was performed to quantify the cancer-related genes expressed in the K14E6 mice. The development and evolution of skin carcinogenesis were assessed through histological analysis to compare cell morphology. RESULTS: Ca 59% of the MCFA were incorporated via esterification into the PC within 12 hours of the reaction. An emulsifier yield used to formulate the NE of 86% was achieved. Nanoemulsions with a particle size of 44 nm were obtained. The curcumin nano-emulsion group had a 91.81% decrease in the tumorigenesis index and a reduction in tumor area of 89.95% compared to the sick group. Histological analysis showed that the group administered with free curcumin developed a microinvasive squamous cell carcinoma, as opposed to the group with nanoemulsion which presented only a slight inflammation. In gene expression, only a significant difference in Cdk4 was observed in the nanoemulsion group.

Palbociclib Effectively Halts Proliferation but Fails to Induce Senescence in Patient-Derived Glioma Stem Cells.

Glioblastoma multiforme is the most aggressive primary brain tumor. Current knowledge suggests that the growth and recurrence of these tumors are due in part to the therapy-resistant glioma stem cell subpopulation, which possesses the ability for self-renewal and proliferation, driving tumor progression. In many cancers, the p16INK4a-CDK4/6-pRb pathway is disrupted in favor of cell cycle progression. In particular, the frequent deregulation of CDK4/6 in cancer positions these kinases as promising targets. Palbociclib, a potent and selective CDK4/6 inhibitor, has been approved by the FDA as a first-line treatment of advanced breast cancer and there is currently interest in evaluating its effect on other cancer types. Palbociclib has been reported to be efficient, not only at halting proliferation, but also at inducing senescence in different tumor types. In this study, we evaluated the effect of this inhibitor on four patient-derived glioma stem cell-enriched cell lines. We found that Palbociclib rapidly and effectively inhibits proliferation without affecting cell viability. We also established that in these cell lines CDK6 is the key interphase CDK for controlling cell cycle progression. Prolonged exposure to Palbociclib induced a senescent-like phenotype characterized by flattened morphology, cell cycle arrest, increased ß-galactosidase activity and induction of other senescent-associated markers. However, we found that after Palbociclib removal cell lines resumed normal proliferation, which implies they conserved their replicative potential. As a whole, our results indicate that in patient-derived glioma stem cell-enriched cell lines, Palbociclib induces a senescent-like quiescence rather than true senescence.

Palbociclib-induced autophagy and senescence in gastric cancer cells.

Targeting cyclin D-CDK4/6 kinase complexes has recently been shown to increase the survival of breast cancer patients with estrogen receptor positive breast tumors. Based on these outcomes, CDK4/6 inhibitors are currently being tested, alone o in combination with other drugs, in the treatment of other malignancies characterized by hyper-activation of cyclin D-CDK4/6 complexes. Nonetheless, a better understanding of the cellular processes that are implemented in response to CDK4/6 inhibition is necessary to expand the therapeutic window and confront the development of drug resistance. Herein, we show that, similar to mammary cells, gastric cancer cells are sensitive to the CDK4/6 inhibitor Palbociclib. Inhibition of CDK4/6 in gastric cancer cells leads to the implementation of cellular senescence. However, whether or not this response is accompanied by induction of autophagy seems to depend on both the pRB and p53 status. In cells retaining expression of both tumor suppressive proteins (AGS gastric cancer cells), exposure to Palbociclib induces senescence and autophagy. However, the simultaneous blockade of CDK4/6 and autophagy in these cells exacerbates the senescence phenotype, an indication that autophagy in these experimental settings represents an adaptive mechanism that promotes cell survival rather than being an effector mechanism of senescence. Interestingly, knocking down p53 resulted in senescence reduction and autophagy blockade, the latter apparently involving a disruption of the degradation of autophagosome cargo.

Inhibitory role of ERß on anterior pituitary cell proliferation by controlling the expression of proteins related to cell cycle progression.

Considering that the role of ERß in the growth of pituitary cells is not well known, the aim of this work was to determine the expression of ERß in normal and tumoral cells and to investigate its implications in the proliferative control of this endocrine gland, by analyzing the participation of cyclin D1, Cdk4 and p21. Our results showed that the expression of ERß decreased during pituitary tumoral development induced by chronic E2 stimulation. The 20 ± 1.6% of normal adenohypophyseal cells expressed ERß, with this protein being reduced in the hyperplastic/adenomatous pituitary: at 20 days the ERß+ population was 10.7 ± 2.2%, while after 40 and 60 days of treatment an almost complete loss in the ERß expression was observed (40 d: 1 ± 0.6%; 60 d: 2 ± 0.6%). The ERα/ß ratio increased starting from tumors at 40 days, mainly due to the loss of ERß expression. The cell proliferation was analyzed in normal and hyperplastic pituitary and also in GH3ß- and GH3ß+ which contained different levels of ERß expression, and therefore different ERα/ß ratios. The over-expression of ERß inhibited the GH3 cell proliferation and expression of cyclin D1 and ERα. Also, the ERß activation by its agonist DPN changed the subcellular localization of p21, inducing an increase in the p21 nuclear expression, where it acts as a tumoral suppressor. These results show that ERß exerts an inhibitory role on pituitary cell proliferation, and that this effect may be partially due to the modulation of some key regulators of the cell cycle, such as cyclin D1 and p21. These data contribute significantly to the understanding of the ER effects in the proliferative control of pituitary gland, specifically related to the ERß function in the E2 actions on this endocrine gland.

1α,25 dihydroxi-vitamin D3 modulates CDK4 and CDK6 expression and localization.

We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21(Waf1/Cip1) and p27(Kip1) expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and ß isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia.

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

Identifying differentially expressed genes and pathways in two types of non-small cell lung cancer: adenocarcinoma and squamous cell carcinoma.

Non-small cell lung carcinoma, NSCLC, accounts for 80-85% of lung cancers. NSCLC can be mainly divided into two types: adenocarcinoma (ADC) and squamous cell carcinoma (SCC). The purpose of our study was to identify and differentiate the pathogenesis of ADC and SCC at the molecular level. The gene expression profiles of ADC and SCC were downloaded from Gene Expression Omnibus under accession No. GSE10245. Accordingly, differentially expressed genes (DEGs) were identified by the limma package in R language. In addition, DEGs were functionally analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. A total of 4124 DEGs were identified, including CDK1, CDK2, CDK4, and SKP2. The DEGs were mainly involved in 16 pathways related to cell proliferation, cell signal transduction and metabolism. We conclude that the molecular mechanisms of ADC and SCC are considerably different, and that they are involved in immune response, cell signal transduction, metabolism, cell division, and cell proliferation. Therefore, the two diseases should be treated differently. This study offers new insight into the diagnosis and therapy of these two types of lung cancer.

Anti-tumour effects of elatol, a marine derivative compound obtained from red algae Laurencia microcladia.

OBJECTIVES: This paper aims to evaluate the anti-tumour properties of elatol, a compound (sesquiterpene) isolated from algae Laurencia microcladia. METHODS: In-vitro and in-vivo anti-tumour properties of elatol were investigated using: MTT assays to assess the cytotoxic effects; flow cytometry analysis to examine the cell cycle and apoptosis; Western blot analysis for determination of the expression of cell cycle and apoptosis proteins; and study of in-vivo tumour growth in mice (C57Bl6 mice bearing B16F10 cells). KEY FINDINGS: Elatol exhibited a cytotoxic effect, at least in part, by inducing cell cycle arrest in the G(1) and the sub-G(1) phases, leading cells to apoptosis. Western blot analysis demonstrated that elatol reduced the expression of cyclin-D1, cyclin-E, cyclin-dependent kinase (cdk)2 and cdk4. A decrease in bcl-xl and an increase in bak, caspase-9 and p53 expression was also observed. In the in-vivo experiment, treatment with elatol was able to reduce tumour growth in C57Bl6 mice. CONCLUSIONS: Elatol promotes a delay in the cell cycle, probably in the G(1)/S transition, activating the apoptotic process and this could be responsible, at least in part, for the in-vivo effects observed. Taken together, the in-vitro and in-vivo experiments suggested that elatol has anti-tumour properties. Further studies should be conducted to clarify the mechanism of action.

EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats.

OBJECTIVES: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. MATERIALS AND METHODS: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. RESULTS: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. CONCLUSIONS: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

Cyclin T1 overexpression induces malignant transformation and tumor growth.

Human PTE Fb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTE Fb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 beta-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.

Can MDM2 and CDK4 make the diagnosis of well differentiated/dedifferentiated liposarcoma? An immunohistochemical study on 129 soft tissue tumours.

BACKGROUND: Well differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS) have been shown to have supernumerary chromosomes with amplified sequences of the MDM2 and CDK4 genes. MDM2 and CDK4 protein overexpression have also been identified in these tumours. AIM: To investigate whether immunohistochemistry (IHC) for MDM2 and CDK4 can be used to diagnose WDLPS and DDLPS. METHODS: IHC for MDM2/CDK4 was carried out on a series of 129 paraffin-embedded lipomatous and non-lipomatous soft tissue tumours. The cases were divided into four groups: WDLPS (n = 19), DDLPS (n = 10), benign adipocytic tumours (BAT) (n = 17), and other mesenquimal tumours (OMT) (n = 83). IHC results were compared in each group and the diagnostic efficacy of the test in identifying WDLPS and DDLPS among the other soft tissue tumours was determined. A percentage of tumour cell positivity was evaluated to better characterise the pattern of tumour immunostaining. RESULTS: Sensitivity and specificity of positive MDM2 and CDK4 immunostainings to identify WDLPS among BAT was 100% and 58.8%, and 68.4% and 88.2%, respectively. When distinguishing DDLPS from OMT, sensitivity and specificity of MDM2 and CDK4 were 90% and 65%, and 70% and 96.3%, respectively. The highest specificity was achieved when a case was considered positive with strong and diffuse immunoreactivity in more than 30% of the neoplastic cells (94.1% and 100%, and 77.1% and 98.8%, respectively). CONCLUSION: Detection of MDM2/CDK4 protein overexpression by IHC can be used by pathologists to diagnose WDLPS and DDLPS. Considering a strong and diffuse immunostaining pattern in most of the neoplastic cells achieves the best results in identifying these tumours.

Molecular mechanism of cell proliferation in rodent uterus during the estrous cycle.

The rodent uterus is a widely studied target tissue for sexual steroid hormone action. The aim of the present study was to assess the molecular mechanism that participates in the initiation of cell proliferation of the rat uterine epithelial cells during the estrus (E)-metestrus (M) transition. Cell proliferation, ERalpha, c-fos, cyclin D1 and D3, cdk4, and cdk6 proteins were assessed in these animals by immunohistochemistry. Estradiol (E(2)) and progesterone (P(4)) plasma levels were assessed by RIA. The results indicate that the glandular epithelium starts to proliferate at 21:00 h on estrus day, and initiates at least 3h before the luminal epithelium does. Fos expression was markedly increased during the afternoon of estrus day, and its increase was in parallel to ERalpha expression. Interestingly, both, cyclin D1 and D3 were abundantly expressed in the luminal and glandular epithelia, and nuclear immunolabelling of cyclin D1 and D3 precedes BrdU incorporation in the cell. cdk4 and cdk6 were localized in the nuclei in both epithelia throughout the studied time course. In addition, cdk4 was more abundant throughout estrus and metestrus days than cdk6. The overall results indicate that ERalpha, Fos and cyclins D1 and D3, cdk4 and cdk6 are expressed in both glandular and luminal epithelia of the rat uterus during the E-M transition. In conclusion, there is a good correlation between sequential expression of these proteins and cell cycle progression in the rat uterine epithelial cells during the estrous cycle. However, the differences observed in the cellular localization, time course of expression and the cellular types that express both cyclins between physiological and pharmacological conditions, demonstrated different mechanisms of regulation and should be due to the complex hormonal milieu during the estrous cycle.

Transcriptional response of peripheral lymphocytes to early fibrosarcoma: a model system for cancer detection based on hybridization signatures.

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer.

Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 status.

MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.

Artepillin C in Brazilian propolis induces G(0)/G(1) arrest via stimulation of Cip1/p21 expression in human colon cancer cells.

Potential chemopreventive agents exist in foods. Artepillin C in Brazilian propolis was investigated for its effects on colon carcinogenesis. We had found that artepillin C was a bioavailable antioxidant, which could be incorporated into intestinal Caco-2 and hepatic HepG2 cells without any conjugation and inhibited the oxidation of intracellular DNA. Artepillin C was then added to human colon cancer WiDr cells. It dose-dependently inhibited cell growth, inducing G(0)/G(1) arrest. The events involved a decrease in the kinase activity of a complex of cyclin D/cyclin-dependent kinase 4 and in the levels of retinoblastoma protein phosphorylated at Ser 780 and 807/811. The inhibitors of the complex, Cip1/p21 and Kip1/p27, increased at the protein level. On the other hand, Northern blotting showed that artepillin C did not affect the expression of Kip1/p27 mRNA. According to the experiments using isogenic human colorectal carcinoma cell lines, artepillin C failed to induce G(0)/G(1) arrest in the Cip1/p21-deleted HCT116 cells, but not in the wild-type HCT116 cells. Artepillin C appears to prevent colon cancer through the induction of cell-cycle arrest by stimulating the expression of Cip1/p21 and to be a useful chemopreventing factor in colon carcinogenesis.