Loss of p14ARF confers resistance to heat shock‐ and oxidative stress‐mediated cell death by upregulating β‐catenin

The p14^ARF is a key tumor suppressor induced mainly by oncogenic stimuli. Although p14^ARF does not seem to respond to DNA damage, there are very few data regarding its role in other forms of stress, such as heat shock (HS) and oxidative stress (OS). Here, we report that suppression of p14^ARF increased resistance to cell death when cells were treated with H₂O₂ or subjected to HS. In this setting, protection from cell death was mediated by elevated levels and activity of β‐catenin, as downregulation of β‐catenin alleviated the protective role of p14^ARF silencing. Moreover, Hsp70 was shown to regulate β‐catenin protein levels by interacting with p14^ARF, suggesting that Hsp70, p14^ARF and β‐catenin form a regulatory network. This novel pathway triggers cell death signals when cells are exposed to HS and OS.     

Peptide‐based tumor inhibitor encoding mitochondrial p14ARF is highly efficacious to diverse tumors

p14^ARF is one of the major tumor suppressors conventionally identified both as the mdm2‐binding molecule restoring p53 function in the nucleus, and as a nucleophosmin‐binding partner inside the nucleolous to stabilize ribosomal RNA. However, its recently reported mitochondrial localization has pointed to novel properties as a tumor suppressor. At the same time, functional peptides are gaining much attention in nanomedicine for their in vivo utility as non‐invasive biologics. We previously reported the p14^ARF‐specific peptide that restored the sensitivity to gefitinib on the gefitinib‐resistant lung cancer cells. Based on the information of this prototype peptide, here we generated the more powerful anti‐tumor peptide “r9‐CatB‐p14 MIS,” which comprises the minimal inhibitory sequence of the mitochondrial targeting p14^ARF protein in combination with the proteolytic cleavage site for cathepsin B, which is activated in various tumor cells, fused with the nine‐polyarginine‐domain for cell penetration, and demonstrated its novel action of regulating mitochondrial function in accordance with localization of endogenous p14^ARF. The p14 MIS peptide showed a potent tumor inhibiton in vitro and in vivo against not only lung cancer cells but also tumor cells of diverse lineages, via modulating mitochondrial membrane potential, with minimal cytotoxicity to non‐neoplastic cells and tissues. Hence, this mitochondrially targeted p14 peptide agent provides a novel basis for non‐invasive peptide‐based antitumor therapeutics.     

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF‐7 and MDA‐MB‐231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI‐Annexin‐V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF‐7 and MDA‐MB‐231 cells in a time‐ and dose‐dependent manner; however, MDA‐MB‐231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell‐cycle arrest, p53 accumulation and activation selectively in MCF‐7 cells. Inspiringly, the PI‐Annexin‐V staining assay and western blot analysis confirmed cell apoptosis executed by caspase‐3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down‐regulation of Bcl‐2 and up‐regulation of BCL2‐associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier‐induced apoptosis was confirmed by pan‐caspase inhibitor, Z‐VAD‐fmk. As expected, the inhibitor decreased Huaier‐induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER‐positive and ER‐negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment. (Cancer Sci 2010; 101: 2375–2383)     

Loss of p21 disrupts p14 ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.     

Lysosomal destabilization and cathepsin B contributes for cytochrome c release and caspase activation in embelin‐induced apoptosis

XIAP is an important antiapoptotic protein capable of conferring resistance to cancer cells. Embelin, the small molecular inhibitor of XIAP, possesses wide spectrum of biological activities with strong inhibition of nuclear factor kappa B and downstream antiapoptotic genes. However, the mechanism of its cell death induction is not known. Our studies using colon cancer cells lacking p53 and Bax suggest that both lysosomes and mitochondria are prominent targets of embelin‐induced cell death. Embelin induced cell‐cycle arrest in G₁ phase through p21, downstream of p53. In the absence of p21, the cells are sensitized to death in a Bax‐dependent manner. The loss of mitochondrial membrane potential induced by embelin was independent of Bax and p53, but lysosomal integrity loss was strongly influenced by the presence of p53 but not by Bax. Lysosomal role was further substantiated by enhanced cathepsin B activity noticed in embelin‐treated cells. p53‐dependent lysosomal destabilization and cathepsin B activation contribute for increased sensitivity of p21‐deficient cells to embelin with enhanced caspase 9 and caspase 3 activation. Cathepsin B inhibitor reduced cell death and cytochrome c release in embelin‐treated cells indicating lysosomal pathway as the upstream of mitochondrial death signaling. Deficiency of cell‐cycle arrest machinery renders cells more sensitive to embelin with enhanced lysosomal destabilization and caspase processing emphasizing its potential therapeutic importance to address clinical drug resistance. © 2009 Wiley‐Liss, Inc.     

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G₂–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐x_L conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

Seliciclib (CYC202, R‐roscovitine) enhances the antitumor effect of doxorubicin in vivo in a breast cancer xenograft model

We sought to determine whether seliciclib (CYC202, R‐roscovitine) could increase the antitumor effects of doxorubicin, with no increase in toxicity, in an MCF7 breast cancer xenograft model. The efficacy of seliciclib combined with doxorubicin was compared with single agent doxorubicin or seliciclib administered to MCF7 cells and to nude mice bearing established MCF7 xenografts. Post‐treatment cells and tumors were examined by cell cycle analysis, immunohistochemistry and real‐time PCR. Seliciclib significantly enhanced the antitumor effect of doxorubicin without additional murine toxicity. MIB1 (ki67) immunohistochemistry demonstrated reduced proliferation with treatment. The levels of p21 and p27 increased after treatment with doxorubicin or seliciclib alone or in combination, compared to untreated controls. However, no changes in p53 protein (DO1, CM1), survivin or p53 phosphorylation (SER¹⁵) were observed in treated tumors compared with controls. In conclusion, the CDK inhibitor seliciclib (R‐roscovitine) enhances the antitumor effect of doxorubicin in MCF7 tumors without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis. © 2008 Wiley‐Liss, Inc.     

Alterations of the p53 and pRB pathways in human astrocytoma

Human astrocytomas are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRB and the p53 pathways. Genetic alterations functionally eliminate pRB and p53 themselves or upstream and/or downstream molecules such as products of theInk4a/ARF locus, p16^Ink4a and p14^ARF. As a result, malignant cells are defective in critical cell cycle and apoptosis regulatory elements contributing to unrelenting tumour growth and invasion. Current research aims to discover effective means of reconstituting p53 and pRB pathway components in an effort to attenuate the aggressive phenotype of astrocytoma.     

A phosphomimetic mutant of RelA/p65 at Ser536 induces apoptosis and senescence: An implication for tumor‐suppressive role of Ser536 phosphorylation

Hundreds of NF‐κB inhibitors have been developed for cancer therapy, but their clinical efficacy is unsatisfactory. Here we show that the phosphorylation activation at Ser536 of RelA/p65 protein, a main subunit in the NF‐κB family, may play a tumor‐suppressive role. In normal colon mucosa, RelA/p65 phosphorylation at Ser536 was increasingly increased with the maturation and apoptotic shedding of epithelial cells, but the phosphorylation at Ser536 was decreased in colon cancer. In colon (HCT116 p53 wt and p53 ?/?), breast (MCF7), and prostate (LNCaP and DU145) cancer cells, a phosphomimetic mutation of RelA/p65 at Ser536 (named p65/S536D) triggered dramatic apoptosis through affecting expression of a wide range of cell death/survival genes, such as Bim, Puma, Noxa, Bcl‐2 and survivin. In HCT116 cells, p65/S536D mutant upregulated Fas, insulted mitochondrial membrane potential, and triggered cleavage and activation of caspase‐3, 7, 8 and 9. A FasL neutralizing antibody (NOK1) prevented cell death induced by the p65/S536D. A pan inhibitor of caspases, Z‐VAD‐FMK (20 μM), blocked caspase‐mediated mitochondrial membrane depolarization. This p65/S536D also triggered senescence in HCT116 cells through a p16‐dependent pathway, but not in MFC7 due to lack of p16. Intratumoral delivery of the p65/S536D effectively suppressed tumor growth in nude mice. Together our data suggest that the phosphorylation of RelA/p65 at Ser536 may confer it a tumor‐suppressive role by inducing apoptosis and senescence, highlighting the importance of discriminating the function and active status of individual active sites in RelA/p65 when NF‐κB inhibitors are considered for targeted therapy of cancer.     

Sensitization to TNF‐induced apoptosis by 1,25‐dihydroxy vitamin D3 involves up‐regulation of the TNF receptor 1 and cathepsin B

The active form of vitamin D₃, 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃), induces caspase‐independent apoptosis in MCF‐7 and T47D breast cancer cells. Before the appearance of apoptotic cells at Day 4 after the addition of 1,25(OH)₂D₃, the MCF‐7 cells are sensitized to the caspase‐mediated apoptosis induced by TNF. We studied the mechanism underlying the cross talk between these 2 distinct death pathways in MCF‐7 and T47D cells. Whereas 1,25(OH)₂D₃ pre‐treatment enhanced TNF‐induced apoptosis of TNF sensitive MCF‐7 cells, it failed to render TNF resistant T47D cells sensitive to this cytokine. Opposing to an earlier report suggesting that cytosolic phospholipase A₂ (cPLA₂) mediates the 1,25(OH)₂D₃‐induced sensitization to TNF, we could not detect any cPLA₂ protein in MCF‐7 cells and its overexpression had no effect on cellular sensitivity to 1,25(OH)₂D₃ or the combination with TNF. The sensitization of MCF‐7 cells to TNF‐induced apoptosis by pre‐treatment with 1,25(OH)₂D₃ may instead be partially explained by an increased surface expression of the TNF receptor 1 (TNF‐R1). In line with this, not only the TNF‐induced activation of caspases and apoptosis but also that of NF‐κB was enhanced by 1,25(OH)₂D₃ pre‐treatment. Furthermore, 1,25(OH)₂D₃ enhanced TNF‐induced NF‐κB activation in T47D cells suggesting that it potentiates TNF signaling in general. Interestingly, the lysosomal protease cathepsin B, which expression is up‐regulated by 1,25(OH)₂D₃, was released from the lysosomes upon TNF treatment, and inhibition of its activity rescued 1,25(OH)₂D₃ treated MCF‐7 cells from TNF‐induced apoptosis. In conclusion, 1,25(OH)₂D₃ may enhance TNF‐induced apoptosis by increasing the expression of both the TNF‐R1 and cathepsin B. © 2001 Wiley‐Liss, Inc. © 2001 Wiley‐Liss, Inc.     

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3‐bromopyruvate

BACKGROUND:

3‐Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3‐bromopyruvate.

METHODS:

The transport of 3‐bromopyruvate by sodium‐coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na⁺)‐coupled, electrogenic transporter for short‐chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3‐bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence‐activated cell‐sorting analysis and colony‐formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis.

RESULTS:

3‐Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na⁺‐coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3‐bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3‐bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3‐bromopyruvate.

CONCLUSIONS:

3‐Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na⁺ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Cancer 2009. © 2009 American Cancer Society.     

NV‐128, a novel isoflavone derivative,induces caspase‐independent cell death through the Akt/mammalian target of rapamycin pathway

BACKGROUND:

Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase‐independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV‐128 as an inducer of cell death through a caspase‐independent pathway.

METHODS:

Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV‐128, and the concentration that caused 50% growth inhibition (GI₅₀) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein‐protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model.

RESULTS:

NV‐128 was able to induce significant cell death in both paclitaxel‐resistant and carboplatin‐resistant EOC cells with a GI₅₀ between 1 μg/mL and 5 μg/mL. Cell death was characterized by chromatin condensation but was caspase‐independent. The activated pathway involved the down‐regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin‐1 followed by nuclear translocation of endonuclease G.

CONCLUSIONS:

The authors characterized a novel compound, NV‐128, which inhibits mTOR and promotes caspase‐independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase‐dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV‐128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society.     

N6‐isopentenyladenosine inhibits cell proliferation and induces apoptosis in a human colon cancer cell line DLD1

N⁶‐isopentenyladenosine (i6A) is a modified nucleoside with a pentaatomic isopentenyl derived from mevalonate that induces inhibition of tumor cell proliferation and apoptosis in several tumor cell lines. In this study, we reported that N⁶‐isopentenyladenosine inhibited the proliferation and promotes apoptosis in DLD1 human colon cancer cells. It suppressed the proliferation of cells through inhibition of DNA synthesis, causing a cell cycle arrest that correlated with a decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a concomitant increase in the levels of cyclin‐dependent kinase inhibitor p21waf and p27kip1. Moreover, it induced apoptosis through an increase in the number of annexin V‐positive cells, a downregulation of antiapoptotic products and caspase‐3 activation. The apoptotic effects of N⁶‐isopentenyladenosine were accompanied by sustained phosphorylation and activation of c‐jun N‐terminal kinase (JNK) that induced phosphorylation of c‐jun. Overall, our data show that JNK, could play an important role in i6A‐mediated apoptosis in DLD1 human colon cancer cells © 2008 Wiley‐Liss, Inc.     

RNAi‐mediated CD73 suppression induces apoptosis and cell‐cycle arrest in human breast cancer cells

Ecto‐5′‐nucleotidase (CD73), a cell surface protein that hydrolyzes extracellular AMP into adenosine and phosphate, is overexpressed in many solid tumors. In this study, we tested the hypothesis that increased CD73 may promote tumor progression by examining the effect of CD73 suppression via RNA interference and CD73 overexpression on tumor growth in vivo and in vitro. Using digitized whole‐body images, plate clone forming assay and TUNEL assay in frozen tissue sections, we found that the cell growth rate was significantly lower in vivo and in vitro after CD73 suppression and late apoptosis was much higher in xenograft tumors developed from the CD73‐siRNA transfected MB‐MDA‐231 clone (P1). By flow cytometry, the P1 cell cycle was arrested in the G0/G1 phase. Moreover, Bcl‐2 was downregulated, while Bax and caspase‐3 were upregulated with CD73 suppression. CD73 inhibitor α,β‐methylene adenosine‐5′‐disphosphate (APCP) functioned similarly with RNAi‐mediated CD73 suppression. In addition, in transfected MCF‐7 cells, we found that CD73 overexpression increased cell viability and promoted cell cycle progression, depending on its enzyme activity. More intriguingly, CD73 overexpression in MCF‐7 breast cancer cells produces a tumorigenic phenotype. We conclude that CD73 plays an important role in breast cancer growth by affecting cell cycle progression and apoptosis. (Cancer Sci 2010; 101: 2561–2569)     

Ratio of Expression of p16INK4a to p14ARF Correlates with the Progression of Non-small Cell Lung Cancer

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16^INK4a and p14^ARF, through the use of independent first exons and shared exons 2 and 3. p16^INK4a is a cyclin‐dependent kinase inhibitor, whereas p14^ARF regulates the cell cycle through a p53 and MDM2–dependent pathway. We have examined the expression of p16^INK4a and p14^ARF using competitive RT‐PCR in 60 non‐small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16^INK4a and p14^ARF were nearly equal or the intensity of the p16^INK4a band slightly exceeded that of p14^ARF in the normal lung tissues (n=60). In 38 tumors the intensity of the p16^INK4a band was similar to or slightly weaker than that of p14^ARF. In 6 tumors the intensity of the p16^INK4a band was weaker than that of p14^ARF. In 15 tumors the intensity of the p14^ARF band was very strong and the p16^INK4a band was barely visible. In only one tumor was the intensity of the p16^INK4a band very strong, while the band of p14^ARF was barely visible. The ratio of the intensity of p16^INK4a to p14^ARF had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II‐IV tumors had significantly lower p16^INK4a to p14^ARF ratios than the p stage I tumors (P=0.036). The T2–4 tumors had significantly lower p16^INK4a to p14^ARF ratios than the T1 tumors (P=0.005). The N1–3 tumors had significantly lower p16^INK4a to p14^ARF ratios than the NO tumors (P=0.014). Our results suggest that the ratio of expression of p16^INK4a to p14^ARFtends to decrease during the progression of NSCLC.     

Stomatin‐like protein 2 inhibits cisplatin‐induced apoptosis through MEK/ERK signaling and the mitochondrial apoptosis pathway in cervical cancer cells

Stomatin‐like protein 2 (STOML2 or SLP‐2) is an oncogenic anti‐apoptotic protein that is upregulated in several types of cancer, including cervical cancer. However, the mechanisms responsible for the SLP‐2 anti‐apoptotic function remain poorly understood. Here, we show that siRNA‐mediated SLP‐2 suppression decreases growth of human cervical cancer HELA and SIHA cells, and increases cisplatin‐induced apoptosis through activation of MEK/ERK signaling and suppression of the mitochondrial pathway. The inhibition of the mitochondrial pathway is mediated by increasing the mitochondrial Ca²⁺ concentration and mitochondrial membrane potential, thereby downregulating p‐MEK and p‐ERK levels, upregulating the Bax/Bcl‐2 ratio, increasing cytochrome C release from mitochondria into the cytosol, and upregulating levels of cleaved‐caspase 9, cleaved‐caspase 3, and cleaved poly ADP‐ribose polymerase (PARP). Overexpression of SLP‐2 using adenovirus‐STOML2 has the opposite effect: it upregulates p‐MEK and p‐ERK and downregulates the Bax/Bcl‐2 ratio and levels of cleaved‐caspase 9 to caspase 9, cleaved‐caspase 3 to caspase 3, and cleaved‐PARP to PARP in cisplatin‐treated cells. These data show that SLP‐2 inhibits cisplatin‐induced apoptosis by activating the MEK/ERK signaling and inhibiting the mitochondrial apoptosis pathway in cervical cancer cells.     

Apoptosis induced by methylene‐blue‐mediated photodynamic therapy in melanomas and the involvement of mitochondrial dysfunction revealed by proteomics

Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene‐blue‐mediated photodynamic therapy (MB‐PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB‐PDT under different conditions, and thereafter subjected to cell viability detection assays. MB‐PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase‐9/caspase‐3 apoptosis pathway. Blocking activation of caspase‐3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. Importantly, proteomics study defining altered protein expression in treated cells suggests the involvement of several mitochondrial proteins playing important roles in electron transfer chain, implying mitochondrial dysfunction during the treatment. Furthermore, a transplantable mouse melanoma model was utilized to estimate the effectiveness of MB‐PDT in vivo. The treated mice displayed decreased tumor size and prolonged survival days, which was associated with enhanced apoptotic cell death. These results, offering solid evidence of the induction of mitochondria‐related apoptosis in tumor cells, reveal new aspects of MB‐PDT having potential to be a palliative treatment of melanoma. (Cancer Sci 2008; 99: 2019–2027)     

TLR3 induction by anticancer drugs potentiates poly I:C‐induced tumor cell apoptosis

Toll‐like receptor 3 (TLR3) has gained recognition as a novel molecular target for cancer therapy because TLR3 activation by its synthetic ligand poly I:C directly causes tumor cell death. Recently, we reported that tumor suppressor p53 increases the expression of TLR3 in several tumor cell lines. Another study also showed that interferon‐α (IFN‐α) up‐regulates TLR3 expression. We thus hypothesized that various anticancer drugs such as p53‐activating reagents and IFNs may potentiate poly I:C‐induced tumor cell death through the up‐regulation of TLR3 expression. Here, we screened several anticancer drugs that, together with poly I:C, effectively cause tumor cell death in colon carcinoma HCT116 cells. We found that the DNA‐damaging reagent 5‐fluorouracil (5‐FU) increased TLR3 mRNA expression and potentiated poly I:C‐induced apoptosis in HCT116 p53^+/+ cells but had only minimal effect in p53^?/? cells, indicating a p53‐dependent pathway. On the other hand, IFN‐α increased poly I:C‐induced apoptosis and the TLR3 mRNA level in HCT116 p53^+/+ and p53^?/? cell lines. Furthermore, the combination of poly I:C, 5‐FU and IFN‐α induced the highest apoptosis in HCT116 p53^+/+ and p53^?/? cells. Taken together, these data suggest that the anticancer drugs increased TLR3 expression and subsequently potentiated poly I:C‐induced apoptosis likely via p53‐dependent and ‐independent pathways. Considering that the p53 status in malignant cells is heterogeneous, this combination approach may provide a highly effective tumor therapy. (Cancer Sci 2010)