Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth

   

The mechanism of suberoylanilide hydroxamic acid in cell growth inhibition involved induction of pRb-2/p130 interaction and nuclear translocation with E2F-4, followed by significant repression in E2F-1 and PCNA nuclear levels, which led to inhibition in DNA synthesis in mammary epithelial cell lines.     

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances taxol-induced cell death in breast cancer

Purpose

The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) enhances taxol-induced antitumor effects against some human cancer cells. The aim of this study is to investigate whether SAHA can enhance taxol-induced cell death against human breast cancer cells and to illustrate the mechanism in detail.

Methods

A panel of eight human breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the inhibitory effects of SAHA, taxol, or their combination by MTT assay. The effects of SAHA with or without taxol on cell cycle distributions, apoptosis, and protein expressions were also examined. The inhibitory effects on tumor growth were characterized in vivo in BALB/c nude mice bearing a breast cancer xenograft model.

Results

Taxol-resistant and multi-resistant breast cancer cells were as sensitive to SAHA as taxol-sensitive breast cancer cells. A dose-dependent synergistic growth inhibition was found in all the tested breast cancer cell lines treated with the SAHA/taxol combinations. The synergetic effect was also observed in the in vivo xenograft tumor model. The cell cycle analysis and apoptosis assay showed that the synergistic effects resulted from enhanced G2/M arrest and apoptosis.

Conclusions

SAHA increased the anti-tumor effects of taxol in breast cancer in vitro and in vivo. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.     

Pretreatment with anti-oxidants sensitizes oxidatively stressed human cancer cells to growth inhibitory effect of suberoylanilide hydroxamic acid (SAHA)

Purpose

Most prostate, colon and breast cancer cells are resistant to growth inhibitory effects of suberoylanilide hydroxamic acid (SAHA). We have examined whether the high oxidative stress in these cells causes a loss of SAHA activity and if so, whether pretreatment with an anti-oxidant can sensitize these cells to SAHA.

Methods

A DNA-Hoechst dye fluorescence measured cell growth and dichlorfluorescein-diacetate (DCF-DA) dye fluorescence measured reactive oxygen species (ROS). Growth inhibitory and ROS-generating activities of SAHA in androgen-treated or untreated LNCaP cells and PC-3 prostate cancer cells, HT-29 and HCT-115 colon cancer cells, MDA-MB231 breast cancer cells and A549 and NCI-H460 lung cancer cells with or without pretreatment with an anti-oxidant Vitamin E was determined. SAHA activity against LNCaP cells treated with another anti-oxidant N-acetyl cysteine (NAC) was also determined. Liquid chromatography–mass spectrometry (LC–MS) was used to determine intracellular SAHA level.

Results

SAHA treatment markedly inhibits LNCaP cell growth, when the cells are at a low ROS level. SAHA is, however, inactive against the same cell line, when the cells are at a high ROS level. A significant decrease in SAHA level was observed in LNCaP cells with high ROS after 24- and 72-h treatment when compared to cells with low ROS. Vitamin E pretreatment that reduces cellular ROS, synergistically sensitizes oxidatively stressed LNCaP, PC-3, HT-29, HCT-115 and MDA-MB231 cells, but not the A-549 and NCI-H460 cells with low ROS to SAHA. NAC treatment also sensitized androgen-treated LNCaP cells to the growth inhibitory effects of SAHA.

Conclusion

Response to SAHA could be improved by combining anti-oxidants such as Vitamin E with SAHA for the treatment of oxidatively stressed human malignancies that are otherwise resistant to SAHA.     

Neoadjuvant chemoradiation compared to neoadjuvant radiation alone and surgery alone for Stage II and III soft tissue sarcoma of the extremities

Introduction

The pan-HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) has previously shown to be a radio-sensitizer to conventional photon radiotherapy (XRT) in pediatric sarcoma cell lines. Here, we investigate its effect on the response of two sarcoma cell lines and a normal tissue cell line to heavy ion irradiation (HIT).

Materials and methods

Clonogenic assays after different doses of heavy ions were performed. DNA damage and repair were evaluated by measuring γH2AX via flow-cytometry. Apoptosis and cell cycle analysis were also measured via flow cytometry. Protein expression of repair proteins, p53 and p21 were measured using immunoblot analysis. Changes of nuclear architecture after treatment with SAHA and HIT were observed in one of the sarcoma cell lines via light microscopy after staining towards chromatin and γH2AX.

Results

Corresponding with previously reported photon data, SAHA lead to an increase of sensitivity to heavy ions along with an increase of DSB and apoptosis in the two sarcoma cell lines. In contrast, in the osteoblast cell line (hFOB 1.19), the combination of SAHA and HIT showed a significant radio-protective effect. Laser scanning microscopy revealed no significant morphologic changes after HIT compared to the combined treatment with SAHA. Immunoblot analysis revealed no significant up or down regulation of p53. However, p21 was significantly increased by SAHA and combination treatment as compared to HIT only in the two sarcoma cell lines - again in contrast to the osteoblast cell line. Changes in the repair kinetics of DSB p53-independent apoptosis with p21 involvement may be part of the underlying mechanisms for radio-sensitization by SAHA.

Conclusion

Our in vitro data suggest an increase of the therapeutic ratio by the combination of SAHA with HIT in infantile sarcoma cell lines.     

Cell cycle and transcriptional control of human myeloid leukemic cells by transforming growth factor beta

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.     

Induction of DNA damage and p21-dependent senescence by Riccardin D is a novel mechanism contributing to its growth suppression in prostate cancer cells in vitro and in vivo

Purpose

Our previous studies had shown that Riccardin D (RD) exhibited cytotoxic effects by induction of apoptosis and inhibition of angiogenesis and topoisomerase II. Here, we reported that apoptosis is not the sole mechanism by which RD inhibits tumor cell growth because low concentrations of RD caused cellular senescence in prostate cancer (PCa) cells.

Methods

Low concentrations of RD were used to treat PCa cells in vitro and in vivo, and senescence-associated β-galactosidase activity, DNA damage response markers, and/or colony-forming ability, cell cycle were analyzed, respectively. We then used siRNA knockdown to identify key factor in RD-triggered cellular senescence.

Results

RD treatment caused growth arrest at G0/G1 phase with features of cellular senescence phenotype such as enlarged and flattened morphology, increased senescence-associated-beta-galactosidase staining cells, and decreased cell proliferation in PCa cells. Induction of cellular senescence by RD occurred through activation of DNA damage response including increases in the phosphor-H2AX, inactivation of Chk1/2, and suppression of repair-related Ku70/86 and phosphor-BRCA1 in PCa cells in vitro and in vivo. Analysis of expression levels of p53, p21^CIP1, p16^INK4a, p27^KIP1, pRb and E2F1 and genetic knockdown of p21^CIP1 demonstrated an important role of p21^CIP1 in RD-triggered cellular senescence.

Conclusions

Involvement of the DNA damage response and p21^CIP1 defines a novel mechanism of RD action and indicates that RD could be further developed as a promising anticancer agent for cancer therapy.     

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells

Purpose

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL.

Methods

Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms.

Results

The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression.

Conclusions

TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.     

The prolyl oligopeptidase inhibitor SUAM-14746 attenuates the proliferation of human breast cancer cell lines in vitro

Background

Prolyl oligopeptidase (POP, EC 3.4.1.26) is a serine peptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We previously reported that POP inhibition suppressed the growth of NB-1 human neuroblastomas cells and KATO III human gastric cancer cells. POP activity is commonly elevated in many cancers, which includes breast cancer. However, the effect of POP inhibition as a candidate breast cancer therapy is unknown.

Methods

The effects of POP inhibition and knockdown on the proliferation of cultured human estrogen receptor-positive (ER+) MCF7 and T47D, and ER-negative (ER?) MDA-MB-231 breast cancer cell lines and the MCF12A non-tumorigenic epithelial cell line were tested by analyzing their influence on cell proliferation (WST-1 assay), cell viability (trypan blue exclusion assay), and cell cycle arrest (cell cycle analysis, cell cycle regulator proteins expression).

Results

POP-specific inhibitors 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thiopropyl-thioprolinal and RNAi-mediated POP knockdown inhibited the proliferation of MCF7 cells without inducing cell death. SUAM-14746-induced growth inhibition was also observed in T47D and MDA-MB-231 cells, but not in MCF12A cells. This growth inhibition was associated with G₁ phase arrest; reduced cyclin D1 and D3, cyclin-dependent kinase 4 (CDK4), E2F1, and retinoblastoma protein (pRb) expression; and increased cyclin-dependent kinase inhibitor 1B (p27^kip1) expression. Moreover, the SUAM-14746-mediated cell cycle arrest of MCF7 cells was associated with increased pRb2/p130 protein expression and an increase in the number of cells in the quiescent G₀ state, as defined by low RNA levels.

Conclusions

SUAM-14746 inhibited breast cancer cell growth in a cytostatic manner without inducing lethality, and POP-specific inhibitors may be an effective treatment against ER+ and ER? breast cancer.

     

Growth-suppressive effect of suberoylanilide hydroxamic acid (SAHA) on human oral cancer cells

Purpose

The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) has been reported to exhibit anticancer activities in various cancer cell types, but as yet there are few reports on the anticancer effects of SAHA in oral squamous cell carcinoma (OSCC)-derived cells and xenograft models.

Methods

The anti-proliferative and apoptotic activities of SAHA were assessed in human HSC-3 and HSC-4 (OSCC)-derived cell lines and JB6 normal mouse skin-derived epidermal cells using histone acetylation, soft agar colony formation, trypan blue exclusion, 4′-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead viability/cytotoxicity and Western blot analyses.

Results

We found that SAHA treatment resulted in hyperacetylation of histones H2A and H3 and a concomitant decrease in the viability of HSC-3 and HSC-4 cells. SAHA also significantly inhibited the neoplastic transformation of JB6 cells treated with TPA, whereas the viability of these cells was not affected by this treatment. Additionally, we found that SAHA suppressed the anchorage-independent growth (colony forming capacity in soft agar) of HSC-3 and HSC-4 cells. DAPI staining, Live/Dead and Western blot analyses revealed that SAHA can induce caspase-dependent apoptosis in HSC-3 and HSC-4 cells. We also found that SAHA treatment led to inhibition of ERK phosphorylation, and that two MEK inhibitors potentiated SAHA-mediated apoptosis. Okadaic acid treatment inhibited SAHA-mediated apoptosis in both the HSC-3 and HSC-4 cell lines, wheras SAHA induced a profound in vivo inhibition of tumor growth in HSC-3 xenografts.

Conclusions

Our results indicate that the ERK signaling pathway may constitute a critical denominator of SAHA-induced apoptosis in OSCC-derived cells and that SAHA may have therapeutic potential for OSCC.

     

TGF-beta induced RBL2 expression in renal cancer cells by down-regulating miR-93

Purpose

TGF-beta can induce G1 arrest via many mechanisms including up-regulating p21, p27, and Rb. However, as the member of Rb family, whether RBL2 is induced by TGF-beta treatment remains exclusive.

Methods

The expression of RBL2 and miR-93 after TGF-beta treatment was determined by quantitative real-time PCR and western blot. The growth of renal cancer cells was determined by CCK-8 assays and cell cycle was determined by PI staining. The binding of miR-93 on RBL2 3′-UTR was determined by double luciferase system.

Results

In renal cancer cells, TGF-beta treatment induced expression of RBL2 in a time- and concentration-dependent manner, and RBL2 mediated TGF-beta induced growth inhibition and cell cycle arrest in renal cancer cells. Furthermore, we found that miR-93 directly targeted RBL2 by binding to its 3′-UTR in renal cancer cells. Over-expression of miR-93 significantly reduced the expression of RBL2, whereas knock down of miR-93 up-regulated the expression of RBL2. More importantly, TGF-beta treatment inhibited miR-93 expression, which resulted in up-regulation of RBL2 after TGF-beta treatment.

Conclusion

TGF-beta induced RBL2 expression through down-regulating miR-93 in renal cancer cells. The newly identified TGF-beta/miR-93/RBL2 signal pathway reveals a new mechanism of TGF-beta induced growth arrest in renal cancer.     

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation

Background

Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.

Methods

We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.

Results

We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.

Conclusions

HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

     

HIV-1 viral protein R downregulates Ebp1 and stabilizes p53 in glioblastoma U87MG cells

Purpose

HIV-1 viral protein R (Vpr) inhibits cell growth and induces apoptosis in a wide range of cancers. However, the mechanism by which Vpr induces cell cycle arrest and apoptosis in GBM cell lines is unclear. The present work was taken to detect the proteins interacted with Vpr in U87MG cells.

Methods

We analyzed the differential expression of proteins between glioblastoma cell U87MG treated with Ad-Vpr and untreated by 2-DE. We used antibody array analysis to analyze the common molecules in the apoptosis of U87MG induced by Vpr.

Results

We analyzed the differential expression of proteins between U87MG cell treated with Ad-Vpr and untreated, and found that proteins related to DNA damage repair or different apoptosis pathways were involved in the G₂ arrest and apoptosis mediated by Vpr. In addition, proliferation-associated protein 2G4 (PA2G4), also known as Ebp1, was down-regulated and p53 was up-regulated in U87MG cells treated with Ad-Vpr.

Conclusions

Our data suggest that Vpr may inhibit Ebp1 to stabilize p53, which in turn leads to G₂ arrest and apoptosis in U87MG cells.     

Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents

Background:

In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated.

Methods:

Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry.

Results:

The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ∼140 kDa, which increased to ∼290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ∼150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21.

Conclusion:

G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs activity of p21 within the complex.     

Plant HDAC inhibitor chrysin arrest cell growth and induce p21 WAF1 by altering chromatin of STAT response element in A375 cells

Background

Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells.

Methods

Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays.

Results

Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21^WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis.

Conclusion

Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (?692 to ?684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21 ^WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK inhibitor p21 ^WAF1 .     

Caspase activation and changes in Bcl-2 family member protein expression associated with E2F-1-mediated apoptosis in human esophageal cancer cells.

The prognosis for patients with esophageal cancer remains poor, prompting the search for new treatment strategies. Overexpression of E2F-1 has been shown to induce apoptosis in several cancer cell types. In the present study, the effect of adenovirus-mediated E2F-1 overexpression on human esophageal cancer cell lines Yes-4 and Yes-6 was evaluated. Cells were treated by mock infection, infection with an adenoviral vector expressing beta-galactosidase (Ad5CMV-LacZ), or E2F-1 (Ad5CMVE2F-1). Western blot analysis confirmed marked overexpression of E2F-1 in Ad5CMVE2F-1-infected cells. Overexpression of E2F-1 resulted in marked growth inhibition and rapid loss of cell viability due to apoptosis, although Yes-6 cells were somewhat more resistant to E2F-1-mediated growth inhibition than Yes-4 cells. Cell cycle analysis revealed that overexpression of E2F-1 led to G2 arrest, followed by apoptotic cell death. p53 expression remained undetectable in both cell lines after E2F-1 overexpression. The apoptosis inhibitor proteins of the Bcl-2 gene family, Bcl-2, Mcl-1, and BcI-XL, decreased at 48 h after infection in Yes-4 cells, but remained unchanged in Yes-6 cells. Levels of retinoblastoma gene product (pRb) declined at 48 h after E2F-1 infection in Yes-4 cells, at which apoptosis predominated, whereas pRb expression remained constant in Yes-6 cells. Expression of p14ARF did not change after E2F-1 infection in either cell line. Involvement of caspase 3 and caspase 6 in E2F-1-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and poly-ADP-ribose polymerase, as well as fragmentation of the caspase 6 substrate, lamin B. These results indicate that the sensitivity of esophageal cancer cells to E2F-1-mediated apoptosis may be related to differential expression of Bcl-2 family member proteins and suggest that the adenovirus-mediated E2F-1 gene therapy may be a promising treatment strategy for the treatment of this disease.     

Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action

Introduction

The present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA).

Methods

KPL-1 cell growth was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21^Cip1/Waf1, cyclin D₁, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet.

Results

CDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 μmol/l and 270 μmol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G₁ fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G₀/G₁ arrest, which involved increased expression of p53 and p21^Cip1/Waf1, and decreased expression of cyclin D₁. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner.

Conclusion

CDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.     

Gene profiling approaches help to define the specific functions of retinoblastoma family in epidermis

The epidermal-specific ablation of Rb gene leads to increased proliferation, aberrant differentiation, and the disengagement of these processes in vivo and in vitro. These differences in phenotype are more severe with the loss of p107, demonstrating the functional compensation between pRb and p107. As p107 and p130 also exert overlapping functions in epidermis, we have generated Rb(F19/F19)K14cre;Rbl2-/- (pRb-;p130-) mice to analyze possible functional redundancies between pRb and p130. The epidermal phenotype was very similar between pRb- and pRb-;p130- mice, suggesting that pRb and p130 activities are not redundant in epidermis. Importantly, we can correlate the proliferation differences with specific changes in gene expression between pRb-, pRb-;p107- and pRb-;p130- primary keratinocytes using microarray analysis, and explain the phenotypes in the context of altered E2F expression and functionality. Our findings support a model in which the distinct retinoblastoma family members, in conjunction with E2F members, play a central role in regulating epidermal homeostasis through specific or overlapping activities.