Sequence­dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action.

Sorafenib (Nexavar, BAY43­9006 or Sora) is the first molecular targeted agent that has exhibited significant therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond well to Sora and novel therapeutic strategies to optimize the efficacy of Sora are urgently required. Plant­based drugs have received increasing attention owing to their excellent chemotherapeutic and chemopreventive activities; they are also well tolerated, non­toxic, easily available and inexpensive. It is well known that certain biologically active natural products act synergistically with synthetic drugs used in clinical applications. The present study aimed to investigate whether a combination therapy with natural phenolic compounds (NPCs), including curcumin (Cur), quercetin (Que), kaempherol (Kmf) and resveratrol (Rsv), would allow a dose reduction of Sora without concomitant loss of its effectiveness. Furthermore, the possible molecular mechanisms of this synergy were assessed. The hepatic cancer cell lines Hep3b and HepG2 were treated with Sora alone or in combination with NPCs in concomitant, sequential, and inverted sequential regimens. Cell proliferation, cell cycle, apoptosis and expression of proteins associated with the cell cycle and apoptosis were investigated. NPCs markedly potentiated the therapeutic efficacy of Sora in a sequence­, type­, NPC dose­ and cell line­dependent manner. Concomitant treatment with Sora and Cur [sensitization ratio (SR)=28], Kmf (SR=18) or Que (SR=8) was associated with the highest SRs in Hep3b cells. Rsv markedly potentiated the effect of Sora (SR=17) on Hep3b cells when administered in a reverse sequential manner. By contrast, Rsv and Que did not improve the efficacy of Sora against HepG2 cells, while concomitant treatment with Cur (SR=10) or Kmf (SR=4.01) potentiated the cytotoxicity of Sora. Concomitant treatment with Sora and Cur or Kmf caused S­phase and G2/M phase arrest of liver cancer cells and markedly induced apoptosis compared with mono­treatment with Sora, Cur or Kmf. Concomitant treatment with Sora and Cur reduced the protein levels of cyclins A, B2 and D1, phosphorylated retinoblastoma and B­cell lymphoma (Bcl) extra­large protein. By contrast, Sora and Cur co­treatment increased the protein levels of Bcl­2­associated X protein, cleaved caspase­3 and cleaved caspase­9 in a dose­dependent manner. In conclusion, concomitant treatment with Sora and Cur or Kmf appears to be a potent and promising therapeutic approach that may control hepatic cancer by triggering cell cycle arrest and apoptosis. Additional studies are required to examine the potential of combined treatment with Sora and NPCs in human hepatic cancer and other solid tumor types in vivo.

Methylferulate from Tamarix aucheriana inhibits growth and enhances chemosensitivity of human colorectal cancer cells: possible mechanism of action.

BACKGROUND: Natural products are valuable sources for anticancer agents. In the present study, methylferulate (MF) was identified for the first time from Tamarix aucheriana. Spectral data were used for identification of MF. The potential of MF to control cell growth, cell cycle, apoptosis, generation of reactive oxygen species (ROS), cancer cell invasion, nuclear factor kappa B (NFkB) DNA-binding activity and proteasomal activities, as well as the enhancement of chemosensitivity in human colorectal cancer cells, were evaluated. The possible molecular mechanism of MF's therapeutic efficacy was also assessed. METHODS: Column chromatography and spectral data were used for isolation and identification of MF. MTT, immunofluorescence, flow cytometry, in vitro invasion, fluoremetry, EIA and Real time qPCR were used to measure antiproliferative, chemo-sensitizing effects and other biochemical parameters. RESULTS: MF showed a dose-dependent anti-proliferative effect on colorectal cancer cells (IC50 = 1.73 - 1.9 mM) with a nonsignificant cytotoxicity toward normal human fibroblast. Colony formation inhibition (P ≤ 0.001, 0.0001) confirmed the growth inhibition by MF. MF arrested cell cycle progression in the S and G2/M phases; induced apoptosis and ROS generation; and inhibited NF-kB DNA-binding activity, proteasomal activities and cell invasion in colorectal cancer cells. MF up-regulated cyclin-dependent kinase inhibitors (p19 INK4D, p21WAF1/CIP1, p27KIP1), pro-apoptotic gene expression (Bax, Bad, Apaf1, Bid, Bim, Smac) and caspases (caspase 2, 3, 6, 7, 8, 9). Moreover, MF down-regulated cyclin-dependent kinases (Cdk1, Cdk2) and anti-apoptotic gene expression (c-IAP-1, c-IAP-2, Bcl2,FLIP). In addition, MF differentially potentiated the sensitivity of colorectal cancer cells to standard chemotherapeutic drugs. CONCLUSION: MF showed a multifaceted anti-proliferative and chemosensitizing effects. These results suggest the chemotherapeutic and co-adjuvant potential of MF.

Growth inhibitory and chemo-sensitization effects of naringenin, a natural flavanone purified from Thymus vulgaris, on human breast and colorectal cancer.

BACKGROUND: Natural products with diverse bioactivities are becoming an important source of novel agents with medicinal potential. Cancer is a devastating disease that causes the death of millions of people each year. Thus, intense research has been conducted on several natural products to develop novel anticancer drugs. METHODS: Chromatographic and spectral techniques were used for the isolation and identification of naringenin (Nar). MTT, flow cytometry, western blotting, Real Time PCR were used to test anticancer and chemosensitizing effects of Nar, cell cycle, apoptosis, and expression of cell cycle, apoptosis, pro-survival and anti-survival-related genes. RESULTS: In the present study, Thymus vulgaris ethanol extract was purified repeatedly to produce several compounds including the known flavanone, Nar which was identified using different spectral techniques. Nar was shown to inhibit both human colorectal and breast cancer cell growth in a dose- and time-dependent manner through cell cycle arrest at S- and G2/M-phases accompanied by an increase in apoptotic cell death. Additionally, Nar altered the expression of apoptosis and cell-cycle regulatory genes by down-regulating Cdk4, Cdk6, Cdk7, Bcl2, x-IAP and c-IAP-2 and up-regulating p18, p19, p21, caspases 3, 7, 8 and 9, Bak, AIF and Bax in both colorectal and breast cancer cells. Conversely, it diminished the expression levels of the cell survival factors PI3K, pAkt, pIκBα and NFκBp65. Moreover, Nar enhanced the sensitivity of colorectal and breast cancer cells to DNA-acting drugs. DISCUSSION: These findings provide evidence that Nar's pro-apoptotic and chemo-sensitizing effects are mediated by perturbation of cell cycle, upregulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes and inhibition of pro-survival signaling pathways. CONCLUSION: In conclusion, Nar might be a promising candidate for chemoprevention and/or chemotherapy of human cancers. However, further studies exploring this therapeutic strategy are necessary.

Valproic acid, an anti-epileptic drug and a histone deacetylase inhibitor, in combination with proteasome inhibitors exerts antiproliferative, pro-apoptotic and chemosensitizing effects in human colorectal cancer cells: underlying molecular mechanisms.

Although the therapeutic efficacy of valproic acid (VPA) has been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical utility. The present study focused on the development of combined molecular targeted therapies using VPA and proteasome inhibitors (PIs: MG132, PI-1 and PR-39) to determine whether this combination of treatments has synergistic anticancer and chemosensitizing effects against colorectal cancer. Furthermore, the potential molecular mechanisms of action of the VPA/PI combinations were evaluated. The effects of VPA in combination with PIs on the growth of colorectal cancer cells were assessed with regard to proliferation, cell cycle, apoptosis, reactive oxygen species (ROS) generation and the expression of genes that control the cell cycle, apoptosis and pro-survival/stress-related pathways. Treatment with combinations of VPA and PIs resulted in an additive/synergistic decrease in colorectal cancer cell proliferation compared to treatment with VPA or PIs alone. The combination treatment was associated with a synergistic increase in apoptosis and in the number of cells arrested in the S phase of the cell cycle. These events were associated with increased ROS generation, pro-apoptotic gene expression and stress-related gene expression. These events were also associated with the decreased expression of anti-apoptotic genes and pro-survival genes. The combination of VPA with MG132 or PI-1 enhanced the chemosensitivity of the SW1116 (29-185­fold) and SW837 (50-620-fold) colorectal cancer cells. By contrast, the combination of VPA/PR-39 induced a pronounced increase in the chemosensitivity of the SW837 (16-54-fold) colorectal cancer cells. These data provide a rational basis for the clinical use of this combination therapy for the treatment of colorectal cancer.

Superior antimitogenic and chemosensitization activities of the combination treatment of the histone deacetylase inhibitor apicidin and proteasome inhibitors on human colorectal cancer cells.

Despite the effectiveness of histone deacetylase inhibitors, proteasome inhibitors and cytotoxic drugs on human cancers, none of these types of treatments by themselves has been sufficient to eradicate the disease. The combination of different modalities may hold enormous potential for eliciting therapeutic results. In the current study, we examined the effects of treatment with the histone deacetylase inhibitor (HDACI) apicidin (APC) in combination with proteasome inhibitors on human colorectal cancer cells. The molecular mechanisms of the combined treatments and their potential to sensitize colorectal cancer cells to chemotherapies were also investigated. Cancer cells were exposed to the agents alone and in combination, and cell growth inhibition was determined by MTT and colony formation assays. HDAC, proteasome and NF-κB activities as well as reactive oxygen species (ROS) were monitored. Cell cycle perturbation and induction of apoptosis were assessed by flow cytometry. The expression of cell cycle/apoptosis- and cytoprotective/stress-related genes was determined by quantitative PCR and EIA, respectively. The potentiation of cancer cell sensitivity to chemotherapies upon APC/PI combination treatment was also studied. The combination of APC and MG132, PI-1 or epoxomicin potently inhibited cancer cell growth, disrupted the cell cycle, induced apoptosis, decreased NF-κB activity and increased ROS production. These events were accompanied by the altered expression of genes associated with the cell cycle, apoptosis and cytoprotection/stress regulation. The combination treatment markedly enhanced the chemosensitivity of colorectal cancer cells (50-3.7 x 10(4)-fold) in a drug-, APC/PI combination- and colorectal cancer subtype-dependent manner. The results of this study have implications for the development of com-binatorial treatments that include HDACIs, PIs and conventional chemotherapeutic drugs, suggesting a potential therapeutic synergy with general applicability to various types of cancers.

Roscovitine synergizes with conventional chemo-therapeutic drugs to induce efficient apoptosis of human colorectal cancer cells.

AIM: To examine the ability of cyclin-dependent kinase inhibitor (CDKI) roscovitine (Rosco) to enhance the antitumor effects of conventional chemotherapeutic agents acting by different mechanisms against human colorectal cancer. METHODS: Human colorectal cancer cells were treated, individually and in combination, with Rosco, taxol, 5-Fluorouracil (5-FU), doxorubicine or vinblastine. The antiproliferative effects and the type of interaction of Rosco with tested chemotherapeutic drugs were determined. Cell cycle alterations were investigated by fluorescence-activated cell sorter FACS analysis. Apoptosis was determined by DNA fragmentation assay. RESULTS: Rosco inhibited the proliferation of tumor cells in a time- and dose-dependent manner. The efficacies of all tested chemotherapeutic drugs were markedly enhanced 3.0-8.42 multiply 10(3) and 130-5.28 multiply 10(3) fold in combination with 5 and 10 microg/mL Rosco, respectively. The combination of Rosco and chemotherapeutic drugs inhibited the growth of human colorectal cancer cells in an additive or synergistic fashion, and in a time and dose dependent manner. Rosco induced apoptosis and synergized with tested chemotherapeutic drugs to induce efficient apoptosis in human colorectal cancer cells. Sequential, inverted sequential and simultaneous treatment of cancer cells with combinations of chemotherapeutic drugs and Rosco arrested the growth of human colorectal cancer cells at various phases of the cell cycle as follows: Taxol/Rosco (G2/M- and S-phases), 5-FU/Rosco (S-phase), Dox/Rosco (S-phase) and Vinb/Rosco (G2/M- and S-phases). CONCLUSION: Since the efficacy of many anticancer drugs depends on their ability to induce apoptotic cell death, modulation of this parameter by cell cycle inhibitors may provide a novel chemo-preventive and chemotherapeutic strategy for human colorectal cancer.

c-myc antisense oligonucleotides sensitize human colorectal cancer cells to chemotherapeutic drugs.

BACKGROUND/AIMS: Overexpression of the c-myc oncogene frequently occurs in both colon tumors and colon carcinoma cell lines. We examined the sensitization of human colorectal cancer cells to chemotherapeutic drugs using c-myc antisense (AS) phosphorothioate oligonucleotides ([S]ODNs). METHODS: Cancer cells were treated with c-myc [S]ODNs, taxol, 5-fluorouracil (5-FU), doxorubicin and vinblastine individually and in combination. The antiproliferative effects, type of interaction between c-myc [S]ODNs and cytotoxic drugs, cell cycle, apoptosis and expression of cell-cycle- and apoptosis-regulatory genes were evaluated. RESULTS: After treatment with c-myc AS[S]ODNs, the growth of cancer cells was markedly inhibited in a dose- and time-dependent manner and the levels of c-myc mRNA and protein were greatly decreased (p < 0.0001). The combinations of c-myc AS[S]ODNs and cytotoxic drugs produced greater growth inhibition of human colorectal cancer cells compared to single treatment with either c-myc AS[S]ODNs (p < 0.006) or cytotoxic drugs (p < 0.0001). These combinations exhibited time- and dose-dependent additive and/or synergistic antiproliferative effects. Cancer cells treated with cytotoxic drugs were growth arrested in the S phase. In contrast, cells treated with either c-myc AS[S]ODNs or by the combination of c-myc AS[S]ODNs and cytotoxic drugs were growth arrested in the G(2)/M and S phases. The combination treatments also exhibited a marked apoptotic effect compared to single treatments. c-myc AS[S]ODN treatment reduced the mRNA levels of Bcl2, BclxL, cdk2, cyclin E1, cdk1 and cyclin B1, while increasing the mRNA levels of p21, p27, bax and caspase-3. CONCLUSION: This two-hit approach may be important in the quest to overcome drug resistance in cancer patients whose tumors carry an overexpressed c-myc gene.

Antisense oligodeoxynucleotide directed against c-myb has anticancer activity and potentiates the antiproliferative effect of conventional anticancer drugs acting by different mechanisms in human colorectal cancer cells.

The C-MYB proto-oncogene encodes a DNA-binding protein with transactivation properties that plays an important regulatory role in cell proliferation and differentiation. Overexpression of C-MYB in colonic tumors compared to normal mucosa suggests that c-myb may play a role in the malignant transformation of colonic mucosa and that inhibition of c-myb expression may suppress, to some extent, the proliferation of neoplastic cells. Complete suppression of tumor cell proliferation may require inhibition of multiple growth-promoting genes. Alternatively, the combination of oncogene-targeted oligodeoxynucleotides (ODNs) with standard cytotoxic agents might represent a useful therapeutic approach to improving cancer treatment. In the present study, we have investigated whether the inhibition of a growth-promoting gene, namely c-myb, affects the sensitivity of human colorectal cancer cells, in vitro, to conventional chemotherapeutic drugs: taxol, 5-fluorouracil, vinblastine and doxorubicin. We show that c-myb antisense phosphorothioate (S) ODN treatment induces growth arrest in the G(1)/G(2) phases of the cell cycle and inhibits cell proliferation in a dose- and time-dependent manner. Also, treatment with c-myb antisense (S)ODN decreases c-myb mRNA and protein expression. A greater inhibition of cell proliferation in vitro was obtained with the combination of c-myb (S)ODN and cytotoxic drugs. The combinations exerted additive and synergistic effects on human colorectal cancer cells. This study suggests that c-myb antisense (S)ODN might be useful in the therapy of colorectal cancer in combination with chemotherapeutic drugs.