Targeting p53 as a promising therapeutic option for cancer by re-activating the wt or mutant p53's tumor suppression.

p53 protein, a product of the TP53 tumor suppressor gene, controls the cellular genome's integrity and is an important regulator of cell cycling, proliferation, apoptosis and metabolism. Mutations of TP53 or inactivation of its gene product are among the first events initiating malignant transformation. The consequent loss of control over the cell cycle, resulting in accelerated cell proliferation and facilitating metabolic reprogramming, gives the initiated (premalignant) cells numerous advantages over healthy cells. Interestingly, p53 status is not only an important marker in cancer diagnosis; it has also become a promising target of personalized therapy. Depending on the TP53 status different therapeutic options have been developed. (Re)-activation of p53 functionality in cancer cells offers promising new alternatives to existing oncological therapies.

Why (multi)targeting of cyclin-dependent kinases is a promising therapeutic option for hormone-positive breast cancer and beyond.

Estrogens, via induction of their specific receptors (e.g., ER-α), regulate cell proliferation, differentiation and morphogenesis in mammary epithelium. Cell-cycle progression is driven by activation of complexes consisting of cyclin-dependent kinases (CDKs) and cyclins, which also modulate the activity of ER-α. Loss of control over the cell-cycle results in accelerated cell division and malignant transformation. Thus, a reciprocal relation exists between estrogen signaling and cell proliferation. Based on these findings, a new concept was developed to reduce ER-α activity and bring the cell cycle in transformed cells to heel. Prevention of ER-α activation and control over the deregulated cell cycle was achieved by supplementation with pharmacological CDK inhibitors alone or in combination with selective antiestrogens.

Differential Potential of Pharmacological PARP Inhibitors for Inhibiting Cell Proliferation and Inducing Apoptosis in Human Breast Cancer Cells.

BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s).

Interactions Between Ataxia Telangiectasia Mutated Kinase Inhibition, Poly(ADP-ribose) Polymerase-1 Inhibition and BRCA1 Status in Breast Cancer Cells.

BACKGROUND: Cells harboring BRCA1/BRCA2 mutations are hypersensitive to inhibition of poly(ADP-ribose) polymerase-1 (PARP-1). We recently showed that interference with PARP-1 activity by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells but not BRCA1-deficient SKBr-3 cells. These unexpected observations prompted speculation that other PARP-1 inhibitor(s) may be more cytotoxic towards SKBr-3 cells. In addition, interference with the DNA damage signaling pathway via (for instance) Ataxia telangiectasia mutated (ATM) kinase inhibition may induce synthetic lethality in DNA repair-deficient breast cancer cells and pharmacological interference with ATM activity may sensitize breast cancer cells to PARP-1 inactivation. METHODS: We determined drug cytotoxicity in human MCF-7 and SKBr-3 breast cancer cells using the CellTiterGLO Luminescent cell viability assay and a Tecan multi-label, multitask plate counter to measure generated luminescence. Changes in cell cycle progression were monitored by flow cytometric measurement of DNA content in cells stained with propidium iodide. RESULTS: Unlike NU1025, AZD2461, a new PARP-1 inhibitor, markedly reduced the numbers of living MCF-7 and SKBr-3 cells. ATM kinase inhibition (CP466722) was also cytotoxic for both MCF-7 and SKBr-3 cells. Furthermore, AZD2461 enhanced the cytotoxicity of CP466722 in both cell lines by inducing apoptosis, and concurrent inhibition of ATM and PARP-1 reduced cell proliferation more strongly than either single treatment. CONCLUSIONS: Our data show that inhibition of PARP-1 by AZD2461 is synthetically lethal for NU1025-resistant MCF-7 and SKBr-3 breast cancer cells. They also indicate that DNA damage signaling is essential for survival of both SKBr-3 and MCF-7 cells, especially after inactivation of PARP-1.

PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells.

Two cellular proteins encoded by the breast and ovarian cancer type 1 susceptibility (BRCA1 and BRCA2) tumor suppressor genes are essential for DNA integrity and the maintenance of genomic stability. Approximately 5-10% of breast and ovarian cancers result from inherited alterations or mutations in these genes. Remarkably, BRCA1/BRCA2-deficient cells are hypersensitive to selective inhibition of poly(ADP-ribose)polymerase 1 (PARP-1), whose primary functions are related to DNA base excision repair; PARP-1 inhibition significantly potentiates the cytotoxicity of various anti-cancer drugs, including inhibitors of topoisomerase I and II. In the present study, we examined the anti-proliferative and pro-apoptotic effects of C-1305, a selective inhibitor of topoisomerase II, on human breast cancer cell lines with different BRCA1 and p53 statuses. BRCA1-competent breast cancer cell lines exhibited different responses to topoisomerase II inhibition. BT-20 cells that express high levels of BRCA1 levels were most resistant to C-1305 than other tested cells. Surprisingly, pharmacological interference with PARP-1 activity strongly inhibited their proliferation and potentiated the efficacy of C-1305 treatment. In contrast, PARP-1 inhibition only weakly affected the proliferation of BRCA1-deficient SKBr-3 cells and was not synergistic with the effects of C-1305. Further experiments revealed that the inhibition of PARP-1 in BT-20 cells caused the accumulation of DNA strand breaks and induced caspase-3 dependent apoptosis. These results seem to indicate that PARP-1 inhibition can potentiate the cytotoxicity of anti-cancer drugs in cancer cells with functional BRCA1 and suggest that mutations in other DNA repair proteins may render cancer cells more sensitive to interference with PARP-1 activity.

The impact of CDK inhibition in human malignancies associated with pronounced defects in apoptosis: advantages of multi-targeting small molecules.

Malignant cells in chronic lymphocytic leukemia (CLL) and related diseases are heterogeneous and consist primarily of long-lived resting cells in the periphery and a minor subset of dividing cells in proliferating centers. Both cell populations have different molecular signatures that play a major role in determining their sensitivity to therapy. Contemporary approaches to treating CLL are heavily reliant on cytotoxic chemotherapeutics. However, none of the current treatment regimens can be considered curative. Pharmacological CDK inhibitors have extended the repertoire of potential drugs for CLL. Multi-targeted CDK inhibitors affect CDKs involved in regulating both cell cycle progression and transcription. Their interference with transcriptional elongation represses anti-apoptotic proteins and, thus, promotes the induction of apoptosis. Importantly, there is evidence that treatment with CDK inhibitors can overcome resistance to therapy. The pharmacological CDK inhibitors have great potential for use in combination with other therapeutics and represent promising tools for the development of new curative treatments for CLL.

Synergistic effects of erlotinib and everolimus on bronchial carcinoids and large-cell neuroendocrine carcinomas with activated EGFR/AKT/mTOR pathway.

BACKGROUND: Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear. MATERIAL AND METHODS: Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting. RESULTS: Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib. CONCLUSIONS: Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted.

Therapeutic intervention by the simultaneous inhibition of DNA repair and type I or type II DNA topoisomerases: one strategy, many outcomes.

Many anticancer drugs reduce the integrity of DNA, forming strand breaks. This can cause mutations and cancer or cell death if the lesions are not repaired. Interestingly, DNA repair-deficient cancer cells (e.g., those with BRCA1/2 mutations) have been shown to exhibit increased sensitivity to chemotherapy. Based on this observation, a new therapeutic approach termed 'synthetic lethality' has been developed, in which radiation therapy or cytotoxic anticancer agents are employed in conjunction with selective inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). Such combinations can cause severe genomic instability in transformed cells resulting in cell death. The synergistic effects of combining PARP-1 inhibition with anticancer drugs have been demonstrated. However, the outcome of this therapeutic strategy varies significantly between cancer types, suggesting that synthetic lethality may be influenced by additional cellular factors. This review focuses on the outcomes of the combined action of PARP-1 inhibitors and agents that affect the activity of DNA topoisomerases.

Can ex vivo evaluation (testing) predict the sensitivity of CLL cells to therapy with purine analogs in conjunction with an alkylating agent? A comparison of in vivo and ex vivo responses to treatment.

Malfunctions in the regulation of apoptosis cause the accumulation of malignant, long-lived B CD19+/CD5+ cells in chronic lymphocytic leukemia (CLL). The primary goal in CLL therapy is to overcome resistance to apoptosis and efficiently trigger programmed cell death in leukemic cells. This study demonstrated that the in vivo responses of malignant cells from CLL patients after administration of purine analogs (cladribine/fludarabine) with cyclophosphamide vary significantly. For comparative purposes, the sensitivity of leukemic cells obtained from the same CLL patients to conventional purine analogs and the selective CDK inhibitor R-roscovitine (ROSC) was determined, with and without the addition of an alkylating agent, prior to the onset of in vivo therapy. The kinetics and rate of spontaneous and drug-induced apoptosis of CLL cells under ex vivo conditions differed significantly between patients, mirroring the variability observed during in vivo treatment. Interestingly, individual patients' leukemic cells were comparably sensitive to the drugs under both conditions. Of the drugs examined, ROSC exerted the highest therapeutic efficacy under ex vivo conditions. Our results indicate that ex vivo testing might be useful for identifying the most potent first-line therapeutic regimen for specific CLL patients and possibly for the design of therapies tailored for individual CLL patients.

The impact of multi-targeted cyclin-dependent kinase inhibition in breast cancer cells: clinical implications.

INTRODUCTION: The progression of the mammalian cell cycle is driven by the transient activation of complexes consisting of cyclins and cyclin-dependent kinases (CDKs). Loss of control over the cell cycle results in accelerated cell division and malignant transformation and can be caused by the upregulation of cyclins, the aberrant activation of CDKs or the inactivation of cellular CDK inhibitors. For these reasons, cell cycle regulators are regarded as very promising therapeutic targets for the treatment of human malignancies. AREAS COVERED: This review covers the structures and anti-breast cancer activity of selected pharmacological pan-specific CDK inhibitors. Multi-targeted CDK inhibitors affect CDKs involved in the regulation of both cell cycle progression and transcriptional control. The inhibition of CDK7/CDK9 has a serious impact on the activity of RNA polymerase II; when its carboxy-terminal domain is unphosphorylated, it is unable to recruit the cofactors required for transcriptional elongation, resulting in a global transcriptional block. Multi-targeted inhibition of CDKs represses anti-apoptotic proteins and thus promotes the induction of apoptosis. Moreover, the inhibition of CDK7 in estrogen receptor (ER)-positive breast cancer cells prevents activating phosphorylation of ER-α. EXPERT OPINION: These diverse modes of action make multi-targeted CDK inhibitors promising drugs for the treatment of breast cancers.

Effect of anti-estrogen combined with roscovitine, a selective CDK inhibitor, on human breast cancer cells differing in expression of ER.

Roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs) reduces numbers of cancer cells in a concentration-dependent manner. At low doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC efficiently inhibits proliferation of human ER-alpha positive MCF-7 breast cancer cells by inducing G/M arrest and concomitantly initiates apoptosis by a p53-dependent pathway. However, the effect of ROSC is much weaker on MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we have examined the action of ROSC on other breast cancer cell lines differing in ER status and confirmed that tamoxifen (TAM) affects the efficacy of this CDK inhibitor. ROSC was effective against all tested breast cancer cell lines, arresting them at G1/S or G2/M transition and inducing apoptosis in SKBR-3 cells. Interestingly, TAM affected all tested cell lines, irrespective of their ER-a status, and in combination with ROSC it enhanced G1 or G2 arrest. Our results provide evidence that ROSC can be combined with antiestrogen therapy and that the mode of ROSC action strongly depends on the cellular context. The effect of TAM on ER-negative cancer cells indicates that TAM also crosstalks with other steroid hormone receptors.

Triazoloacridone C-1305 abrogates the restriction checkpoint in cells lacking functional p53 and promotes their accumulation in the G2/M phase of the cell cycle.

Triazoloacridone C-1305, a new topoisomerase II inhibitor, exhibits potent cytostatic activity toward various tumours under in vitro and in vivo conditions. Interestingly, mouse cells lacking poly(ADP-ribose) polymerase-1 are much more sensitive to C-1305 than their normal counterparts. In the present study we tested the hypothesis that the functional status of p53 in tumour cells might have an impact on the efficiency of C-1305 in experiments with both p53-deficient human HL-60 promyelocytic leukemia cells and human MCF-7 breast cancer cells harboring a functional p53 pathway. Exposure of both cancer cell lines to C-1305 reduced the number of viable cells in a time- and concentration-dependent manner. Remarkably, however, HL-60 cells were much more strongly affected than MCF-7 cells. Measurements of DNA concentrations in single cells revealed that C-1305 arrested the tested cancer cells at the G/M transition. Analysis of the cell cycle and apoptosis regulators revealed that C-1305 strongly elevated phosphorylation of CDK1 at the inhibitory sites (Thr14/Tyr15) in HL-60 cells. Furthermore, C-1305 increased phosphorylation of pRb protein and CDK2 at Thr160 in HL60 cells, but not in MCF-7 cells. These observations suggest that C-1305 abrogates the restriction checkpoint and promotes G1/S transition in cells lacking functional p53.

Roscovitine, a small molecule CDK inhibitor induces apoptosis in multidrug-resistant human multiple myeloma cells.

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential against rapidly dividing cancers and malignancies, characterized by the accumulation of transformed cells due to deregulation of apoptosis, such as multiple myeloma. In the present study we addressed the possibility that pharmacological CDK inhibitors like Roscovitine (ROSC) may be effective against human multiple myeloma cells that have acquired resistance to doxorubicin (DOX). For this purpose we selected an experimental model of human multiple myeloma-sensitive (RPMI-8226s) and doxorubicin-resistant (RPMI-8226(DOX40)) cell lines. Exposure of RPMI-8826 cells to ROSC markedly increased the proportion of hypoploid cells, representing cells undergoing apoptosis, in both sensitive and resistant cells. Unlike ROSC, DOX at high dosage did not elevate the apoptosis rate in the RPMI-8226(DOX40) cell line. Our results show that ROSC has the capacity to induce apoptosis in the RPMI-8226(DOX40) cells overexpressing the P-gp glycoprotein. Since ROSC not only inhibits cell cycle-related CDKs but also negatively regulates kinases involved in the regulation of transcription, its administration to quiescent multidrug-resistant cells might be advantageous. Inhibition of transcription of pro-survival genes such as BCL2 and MCL-1 as well as destabilization of survivin seems to improve its therapeutic efficacy.

Tamoxifen enhances the anti-proliferative effect of roscovitine, a selective cyclin-dependent kinase inhibitor, on human ER-positive human breast cancer cells.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, can arrest human ER-positive MCF-7 breast cancer cells in the G2 phase of the cell cycle and concomitantly induce apoptosis. The observed effects of ROSC were diminished in MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we decided to test whether combining ROSC with anti-estrogen therapy would modulate the efficacy of ROSC action. Exposure of MCF-7 cells to tamoxifen (TAM) for 24 h decreased the number of living cells by approximately 10%. This was associated with a ca. 25% increase in the G1 cell population and reduction in the proportion of S-phase cells. Unlike TAM, estrogen had very weak effects on the cell cycle progression of MCF-7 cells within 24 h. The proliferation-promoting effect of estrogen did not become evident until cultivation of cells for 48 h. Addition of estrogen to MCF-7 cells 1 h prior to TAM administration abolished the anti-estrogen-induced G1 arrest. Simultaneous treatment of MCF-7 cells with ROSC and TAM strongly enhanced the anti-proliferative effect of ROSC. This was potentiated after co-treatment with estrogen. These results clearly indicate that the efficacy of treating ER-positive breast cancers by ROSC can be enhanced by combined application of antiestrogens.

Combining an FPTase inhibitor with cisplatin facilitates induction of apoptosis in human A549 lung cancer cells.

Despite great efforts to develop efficacious curative treatments, the prognosis for lung cancer patients is poor. In the present study we compared the effects of cisplatin (CP), a strong DNA damaging compound, with those of roscovitine (ROSC), a selective inhibitor of cyclin-dependent kinases (CDKs), on wt p53-positive human A549 lung adenocarcinoma cells harboring a mutated K-RAS gene. Asynchronously growing A549 cells were relatively resistant to CP treatment for 24 h, but after exposure to CP at sufficiently high doses (> or = 20 microM) an accumulation of S-arrested cells was observed. However, after post-incubation of CP-treated cells in a drug-free medium for a further 48 h the number of living cells was markedly reduced. Combining CP with L-744,832, a small molecule FPTase inhibitor (FTI), slightly enhanced its anti-proliferative effect. Interestingly, FTI sensitized A549 cells to CP-induced apoptosis. ROSC inhibited A549 cells at the G/M transition, resulting in a marked decrease in the number of viable cells within 24 h, and prolonged treatment with ROSC for 48 h reduced the frequency of living cells by inducing apoptosis. The effects of ROSC (unlike those of CP) were more strongly enhanced by inhibition of the Ras protein processing pathway. Our preliminary results indicate that functional p53 contributes to the outcome of the therapy in human A549 cells by certain anti-cancer drugs.

Roscovitine, a selective CDK inhibitor, reduces the basal and estrogen-induced phosphorylation of ER-α in human ER-positive breast cancer cells.

Roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrests human estrogen receptor-α (ER-α) positive MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis via a p53-dependent pathway. The effect of ROSC is markedly diminished in MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we decided to examine whether ROSC has any effect on the functional status of the ER-α transcription factor. Exposure of MCF-7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 in a concentration and time-dependent manner. This inhibition of site-specific modification of CDK7 at Ser164/170 prevented phosphorylation of RNA polymerase II and reduced basal phosphorylation of ER-α at Ser118 in non-stimulated MCF-7 cells (resulting in its down-regulation). In MCF-7 cells, estrogen induced strong phosphorylation of ER-α at Ser118 but not at Ser104/Ser106. ROSC prevented this estrogen-promoted activating modification of ER-α. Furthermore, we sought to determine whether the activity of ROSC could be enhanced by combining it with an anti-estrogen. Tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affected breast cancer cell lines irrespective of their ER status. In combination with ROSC, however, it had a different impact, enhancing G(1) or G(2) arrest. Our results indicate that ROSC prevents the activating phosphorylation of ER-α and that its mode of action is strongly dependent on the cellular context. Furthermore, our data show that ROSC can be combined with anti-estrogen therapy. The inhibitory effect of TAM on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors.

Promotion of apoptosis in cancer cells by selective purine-derived pharmacological CDK inhibitors: one outcome, many mechanisms.

The deregulation of apoptosis and the cell cycle are important steps in the onset of cancer, giving cells unlimited reproductive potential and increasing their likelihood of survival. The cell cycle is an essential and tightly regulated four-stage process that effects the accurate duplication and transmission of genetic content to cells' progeny. Cyclin-dependent kinases (CDKs) are key elements of the mammalian cell cycle machinery. Their activity is normally regulated via cyclin binding, phosphorylation events, and interactions with endogenous CDK inhibitors. Malfunctions in the control of the cell cycle can be specifically countered using pharmacological CDK inhibitors. Importantly, CDK inhibitors are very effective against both rapidly dividing and quiescent cancer cells; this is particularly relevant in the treatment of malignancies such as chronic lymphatic leukemia (CLL) and multiple myeloma (MM) that exhibit both a low mitotic index and apoptotic defects. The high efficacy of pharmacological CDK inhibitors against CLL and MM is attributable to their ability to eliminate leukemic cells by apoptosis. Indeed, not only do pharmacological CDK inhibitors block cell cycle progression; they also promote apoptosis and thereby destroy irrevocably malignant cells. This article focuses on the impact of inhibiting individual cellular CDKs on apoptosis. We discuss in detail the molecular mechanisms by which CDK inhibitors are able to bypass chemoresistance in tumor cells and trigger apoptosis. Remarkably, recent findings suggest that the pharmacological utility of CDK inhibitors may not be restricted to the treatment of cancer: some may be efficacious in the treatment of patients with neurodegenerative and cardiovascular diseases.

Interference with ER-α enhances the therapeutic efficacy of the selective CDK inhibitor roscovitine towards ER-positive breast cancer cells.

In recent years many risk factors for the development of breast cancer that are linked to estrogens have been identified, and roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, has been shown to be an efficient inhibitor of the proliferation of human breast cancer cells. Therefore, we have examined the possibility that interference with estrogen signaling pathways, using tamoxifen (TAM), a selective estrogen receptor modulator (SERM), could modulate the efficacy of treatment with ROSC. In conjunction with TAM, ROSC exhibited enhanced anti-proliferative activity and CDK inhibition, particularly in estrogen-dependent MCF-7 cells. The interaction between both drugs was synergistic. However, in ER-α-negative cells the interaction was antagonistic. Exposure of MCF-7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 at Ser(164/170). This in turn prevented the phosphorylation of the carboxyl-terminal repeat domain of RNA Polymerase II and ER-α at Ser(118), resulting in the down-regulation of the latter. Concomitantly, wt p53 was strongly activated by phosphorylation at Ser(46). Our results demonstrate that ROSC negatively affects the functional status of ER-α, making it potentially useful in the treatment of estrogen-dependent breast cancer cells.

Whether to target single or multiple CDKs for therapy? That is the question.

Complexes consisting of cyclin-dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK-cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16(INK4A) or p27(Kip1), and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad-spectrum unspecific, pan-specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan-specific and highly selective CDK inhibitors in therapy.

Reconstitution of human MCF-7 breast cancer cells with caspase-3 does not sensitize them to action of CDK inhibitors.

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.