Carvedilol protects against apoptotic cell death induced by cisplatin in renal tubular epithelial cells

Cisplatin is a highly effective chemotherapeutic drug; however, its use is limited by nephrotoxicity. Studies showed that the renal injury produced by cisplatin involves oxidative stress and cell death mediated by apoptosis and necrosis in proximal tubular cells. The use of antioxidants to decrease cisplatin-induced renal cell death was suggested as a potential therapeutic measure. In this study the possible protective effects of carvedilol, a beta blocker with antioxidant activity, was examined against cisplatin-induced apoptosis in HK-2 human kidney proximal tubular cells. The mitochondrial events involved in this protection were also investigated. Four groups were used: controls (C), cisplatin alone at 25 μM (CIS), cisplatin 25 μM plus carvedilol 50 μM (CV + CIS), and carvedilol alone 50 μM (CV). Cell viability, apoptosis, caspase-9, and caspase-3 were determined. Data demonstrated that carvedilol effectively increased cell viability and minimized caspase activation and apoptosis in HK-2 cells, indicating this may be a promising drug to reduce nephrotoxicity induced by cisplatin.     

Possible involvement of oxidative stress in cisplatin-induced apoptosis in LLC-PK1 cells

Use of cisplatin, a chemotherapeutic agent, is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1, 2, 3, 8, and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced, and except for caspase 1, all the other caspases tested were activated. Z-VAD, the broad-spectrum inhibitor of caspases, prevented caspase activation and apoptosis, but not c-myc and p53 induction. On the other hand, N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity.     

Penta-O-galloyl-β-D-glucose attenuates cisplatin-induced nephrotoxicity via reactive oxygen species reduction in renal epithelial cells and enhances antitumor activity in Caki-2 renal cancer cells

Cisplatin shows limited therapeutic efficacy due to serious side effects such as nephrotoxicity and hepatotoxicity. In the present study, we demonstrate that 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) has protective effects against cisplatin-induced cytotoxicity and apoptosis in normal human primary renal epithelial cells (HRCs) while showing synergistic effect against cisplatin-induced cell death in human Caki-2 renal cancer cells. PGG significantly blocked cisplatin-mediated cytotoxicity and reduced cisplatin-induced sub-G1 accumulation in HRCs. Consistently, PGG reduced the number of apoptotic cell populations by TdT-mediated dUTP nick end labeling (TUNEL) and Live/Dead assays in cisplatin-treated HRCs. Furthermore, PGG suppressed PARP cleavage and caspase-3 activation, cytochrome c release, up-regulation of bax and p53 in cisplatin-treated HRCs. Moreover, PGG attenuated reactive oxygen species (ROS) production mediated by cisplatin treatment, suggesting that PGG prevented cisplatin-induced apoptosis by inhibiting ROS generation in HRCs. Notably, PGG significantly enhanced cytotoxicity and PARP cleavage in cisplatin-treated Caki-2 renal cancer cells. Combination Index (CI) revealed synergism between PGG and cisplatin in Caki-2 cells. Taken together, our findings suggest the dual effects of PGG as a protective supplement against cisplatin-induced toxicity in normal renal cells and a combination chemotherapeutic drug with cisplatin in renal cancer cells.     

Protective role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cisplatin-induced nephrotoxicity

Although cisplatin is widely used as an anti-cancer agent, its use is significantly limited because of its tendency to induce nephrotoxicity through poorly understood mechanisms. NAD(P)H:quinone oxidoreductase 1 (NQO1) is well known to regulate ROS generation. The purpose of this study was to investigate whether NQO1 modulates cisplatin-induced renal failure associated with NADPH oxidase (NOX)-derived ROS production in an animal model. NQO1^−/− mice were treated with cisplatin (18 mg/kg) and renal function, oxidative stress, and tubular apoptosis were assessed. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In accordance with these results, the cellular NADPH/NADP ratio and NOX activity were markedly increased in the kidneys of NQO1^−/− mice compared to NQO1^+/+ mice. In addition, activation of NQO1 by βL treatment significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis. This study demonstrates that NQO1 protects cells against renal failure induced by cisplatin, and that this effect is mediated by decreased NOX activity via cellular NADPH/NADP modulation. These results provide convincing evidence that NQO1 might be beneficial for ameliorating renal failure induced by cisplatin.     

Protective role of phosphatidylcholine against cisplatin-induced renal toxicity and oxidative stress in rats

Although cisplatin is widely used in the treatment of cancers, clinical use of cisplatin is limited due to its nephrotoxicity. Pathophysiological mechanism of cisplatin-induced renal toxicity is a complex process and has not been fully understand. Reactive oxygen species (ROS) and oxidative stress have been presumed to be involved in this damage process. Phosphatidylcholine (PC) has antioxidant effect and prevents oxidative stress. Therefore, the present study aimed to investigate potential protective effects of PC on cisplatin-induced renal damage in rat. We examined the protective effects of PC on cisplatin-induced renal damage by assessment of serum creatinine, BUN, lipid peroxidation, total glutathione, glutathione peroxidase activity, catalase activity, superoxide dismutase activity and histophathological changes. PC ameliorated cisplatin-induced increases in serum creatinine, urea and oxidative stress. PC also decreased tubular degeneration and hypertrophy of glomeruli. PC may have a protective effect against cisplatin-induced nephrotoxicity in rats via enhancing antioxidant enzyme activity.     

The ameliorative effect of 23-hydroxytormentic acid isolated from Rubus coreanus on cisplatin-induced nephrotoxicity in rats

Previously, the authors demonstrated that the triterpenoid glycoside niga-ichigoside F? (NIF?) and its aglycone 23-hydroxytormentic acid (23-HTA) isolated from the unripe fruits of Rubus coreanus (Rosaceae) ameliorate cisplatin-induced toxicity in renal epithelial LLC-PK? cells. In the present study, the nephroprotective effects of NIF? and 23-HTA were investigated in Sprague-Dawley rats with acute renal injury induced by a single intraperitoneal (i.p.) injection of cisplatin (7 mg/kg). Pretreatment with 23-HTA (10 mg/kg/d, per os (p.o.)) significantly reduced cisplatin-induced elevations in blood urea nitrogen (BUN) and serum creatinine level, whereas NIF? (10 mg/kg, p.o.) slightly reduced these levels. In addition, pretreatment with 23-HTA prevented cisplatin-induced hydroxyl radical generation, malondialdehyde (MDA) production, glutathione (GSH) depletion, and cisplatin-induced changes in the activities of oxidant and antioxidant enzymes in rat renal tissues. In addition, histopathological examinations showed that 23-HTA pretreatment reduced cisplatin-induced acute tubular necrosis and histological changes. In contrast, NIF? was found to have a slight or no influence on cisplatin-induced oxidative enzymes and acute tubular necrosis. Taken together, these results suggest that protective effect of 23-HTA pretreatment on cisplatin-induced renal damage is associated with the attenuation of oxidative stress and the preservation of antioxidant enzymes.     

The dimethylthiourea-induced attenuation of cisplatin nephrotoxicity is associated with the augmented induction of heat shock proteins

Dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, affords protection against cisplatin (CDDP)-induced acute renal failure (ARF). Since the suppression of oxidative stress and the enhancement of heat shock proteins (HSPs) are both reported to protect against CDDP-induced renal damage, we tested whether increased HSP expression is involved in the underlying mechanisms of the DMTU-induced renal protection. We examined the effect of DMTU treatment on the expression of HSPs in the kidney until day 5 following a single injection of CDDP (5 mg/kg BW). DMTU significantly inhibited the CDDP-induced increments of serum creatinine, the number of 8-hydroxyl-2′-deoxyguanosine (8-OHdG)- and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive tubular cells, and tubular damage score (p < 0.05). CDDP significantly increased renal abundances of HO-1, HSP60, HSP72 and HSP90 at days 1, 3, and 5. DMTU significantly augmented only the expression of HSP60 expression mainly in the cytoplasm of the proximal tubular cells at days 1 and 3 in CDDP-induced ARF. DMTU also inhibited the CDDP-induced increment of Bax, a pro-apoptotic protein, in the fraction of organelles/membranes at day 3. The findings suggest that DMTU may afford protection against CDDP-induced ARF, partially through the early induction of cytoplasmic HSP60, thereby preventing the Bax-mediated apoptosis in renal tubular cells.     

Oxidative stress-driven mechanisms of nordihydroguaiaretic acid-induced apoptosis in FL5.12 cells

Nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) enzyme inhibitor, induces apoptosis independently of its activity as a LOX inhibitor in murine pro-B lymphocytes (FL.12 cells) by a mechanism that is still not fully understood. Glutathione depletion, oxidative processes and mitochondrial depolarization appear to contribute to the apoptosis induced by NDGA. The current data demonstrate that NDGA (20 microM)-induced apoptosis in FL5.12 cells is partially protected by N-acetylcysteine (NAC) (10 mM) and dithiothreitol (DTT) (500 microM) pretreatment, confirming a role for oxidative processes. In addition, the treatment of FL5.12 cells with NDGA led to an increase in phosphorylation and activation of the MAP kinases ERK, JNK and p38. Although pretreatment with ERK inhibitors (PD98059 or U0126) abolished ERK phosphorylation in response to NDGA, neither inhibitor had any effect on NDGA-induced apoptosis. SP600125, a JNK inhibitor, did not have any effect on NDGA-induced phosphorylation of JNK nor apoptosis. Pretreatment with the p38 inhibitor SB202190 attenuated NDGA-induced apoptosis by 30% and also abolished p38 phosphorylation, compared to NDGA treatment alone. NAC, but not DTT, also decreased the phosphorylation of p38 and JNK supporting a role for oxidative processes in activating these kinases. Neither NAC nor DTT blocked the phosphorylation of ERK suggesting that this activation is not related to oxidative stress. The release of cytochrome c and activation of caspase-3 induced by NDGA were inhibited by NAC. SB202190 slightly attenuated caspase-3 activation and had no effect on the release of cytochrome c. These data suggest that several independent mechanisms, including oxidative reactions, activation of p38 kinase and cytochrome c release contribute to NDGA-induced apoptosis.     

Knockdown of NAPA using short-hairpin RNA sensitizes cancer cells to cisplatin: Implications to overcome chemoresistance

Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA—a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking—protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-α and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.     

Cisplatin-induced apoptosis by translocation of endogenous Bax in mouse collecting duct cells

cis-platinum(II) (cis-diammine dichloroplatinum; cisplatin) is a potent antitumor compound that is widely used for the treatment of many malignancies. An important side-effect of cisplatin is nephrotoxicity, which results from injury to renal tubular epithelial cells and can be manifested as either acute renal failure or a chronic syndrome characterized by renal electrolyte wasting. Recently, apoptosis has been recognized as an important mechanism of cell death mediating the antitumor effect of cisplatin. This study was undertaken to examine the mechanisms of cell death induced by cisplatin in M-1 cells, which were derived from the outer cortical collecting duct cells of SV40 transgenic mice. Treatment of M-1 cells with high concentrations of cisplatin (0.5 and 1 mM) for 2 hr led to necrotic cell death, whereas a 24-hr treatment with 5-20 microM cisplatin led to apoptosis. Antioxidants protected against cisplatin-induced necrosis, but not apoptosis, indicating that reactive oxygen species play a role in mediating necrosis but not apoptosis induced by cisplatin and that the mechanism of cell death induced by cisplatin is concentration dependent. The low concentrations of cisplatin, which induced apoptosis in M-1 cells, did not affect the expression levels of Bcl-2-related proteins and did not activate c-Jun NH2-terminal kinase (SAPK/JNK). Cisplatin induced the translocation of endogenous Bax from the cytosolic to the membrane fractions and, subsequently, the release of cytochrome c. Overexpression of Bcl-2 blocked cisplatin-induced apoptosis and Bax translocation. These observations suggest that the subcellular redistribution of Bax is a critical event in the apoptosis induced by cisplatin.     

Signalling mechanisms involved in renal pathological changes during cisplatin-induced nephropathy

Context

Cisplatin, a coordination platinum complex, is used as a potential anti-neoplastic agent, having well recognized DNA-damaging property that triggers cell-cycle arrest and cell death in cancer therapy. Beneficial chemotherapeutic actions of cisplatin can be detrimental for kidneys.

Background

Unbound cisplatin gets accumulated in renal tubular cells, leading to cell injury and death. This liable action of cisplatin on kidneys is mediated by altered intracellular signalling pathways such as mitogen-activated protein kinase (MAPK), extracellular regulated kinase (ERK), or C- Jun N terminal kinase/stress-activated protein kinase (JNK/SAPK). Further, these signalling alterations are responsible for release and activation of tumour necrosis factor (TNF-α), mitochondrial dysfunction, and apoptosis, which ultimately cause the renal pathogenic process. Cisplatin itself enhances the generation of reactive oxygen species (ROS) and activation of nuclear factor-κB (NF-κB), inflammation, and mitochondrial dysfunction, which further leads to renal apoptosis. Cisplatin-induced nephropathy is also mediated through the p53 and protein kinase-Cδ (PKCδ) signalling pathways.

Objective

This review explores these signalling alterations and their possible role in the pathogenesis of cisplatin-induced renal injury.     

Serum thymic factor, FTS, attenuates cisplatin nephrotoxicity by suppressing cisplatin-induced ERK activation

Serum thymic factor (FTS), a thymic peptide hormone, has been reported to attenuate the bleomycin-induced pulmonary injury and also experimental pancreatitis and diabetes. In the present study, we investigated the effect of FTS on cis-diamminedichloroplatinum II (cisplatin)-induced nephrotoxicity. We have already demonstrated that cephaloridine, a nephrotoxic antibiotic, leads to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney, which probably contributes to cephaloridine-induced renal dysfunction. The aim of this study was to examine the effect of cisplatin on ERK activation in the rat kidney and also the effect of FTS on cisplatin-induced nephrotoxicity in rats. In vitro treatment of LLC-PK1 cells with FTS significantly ameliorated cisplatin-induced cell injury. Treatment of rats with intravenous cisplatin for 3 days markedly induced renal dysfunction and increased platinum contents in the kidney cortex. An increase in pERK was detected in the nuclear fraction prepared from the rat kidney cortex from days 1 to 3 after injection of cisplatin. FTS suppressed cisplatin-induced renal dysfunction and ERK activation in the kidney. FTS did not influence any Pt contents in the kidney after cisplatin administration. FTS has been shown to enhance the in vivo expression of heat shock protein (HSP) 70 in the kidney cortex. The beneficial role of FTS against cisplatin nephrotoxicity may be mediated in part by HSP70, as suggested by its up-regulation in the kidney cortex treated with FTS alone. Our results suggest that FTS participates in protection from cisplatin-induced nephrotoxicity by suppressing ERK activation caused by cisplatin.     

Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation

We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.     

Concomitant lansoprazole ameliorates cisplatin-induced nephrotoxicity by inhibiting renal organic cation transporter 2 in rats

Cisplatin is used widely for the treatment of multiple solid tumors. Cisplatin-induced nephrotoxicity is caused by renal accumulation of cisplatin via human organic cation transporter 2 (hOCT2). As lansoprazole, a proton pump inhibitor, is known to inhibit hOCT2 activity, lansoprazole might ameliorate cisplatin-induced nephrotoxicity. A previous study showed that concomitant lansoprazole administration ameliorated nephrotoxicity in patients receiving cisplatin. However, the detailed mechanism remains to be clarified. In the present study, the drug–drug interaction between lansoprazole and cisplatin was examined using hOCT2-expressing cultured cells and rat renal slices. Moreover, the effect of lansoprazole on cisplatin-induced nephrotoxicity and the pharmacokinetics of cisplatin in rats was investigated. In the uptake study, lansoprazole potently inhibited the uptake of cisplatin in hOCT2-expressing cultured cells and rat renal slices. The in vivo rat study showed that concomitant lansoprazole significantly ameliorated cisplatin-induced nephrotoxicity and reduced the renal accumulation of platinum up to approximately 60% of cisplatin alone at 72 h after cisplatin intraperitoneal administration. Furthermore, the renal uptake of platinum at 3 min after intravenous cisplatin administration in rats with cisplatin and lansoprazole decreased to 78% of rats with cisplatin alone. In addition, there was no significant difference in the plasma platinum concentration between rats treated with and without lansoprazole at 3 min after cisplatin intravenous administration. These findings suggested that concomitant lansoprazole ameliorated cisplatin-induced nephrotoxicity by inhibiting rOCT2-mediated cisplatin uptake in rats, thus decreasing cisplatin accumulation in the kidney. The present findings provided important information for the establishment of novel protective approaches to minimize cisplatin-induced nephrotoxicity.     

Mitochondrial/lysosomal toxic cross-talk plays a key role in cisplatin nephrotoxicity

1. Cisplatin is widely used chemotherapeutic agent for the treatment of several human malignancies. Dose-related nephrotoxicity is the major adverse effect of cisplatin. The cellular and molecular mechanisms behind the cisplatin nephrotoxicity have not yet been completely understood.

2. In this study, cytotoxic effect of cisplatin on renal proximal tubular (RPT) cells was evaluated. Our results showed that cytotoxic action of cisplatin on RPT cells is mediated by reactive oxygen species (ROS) formation, decline of mitochondrial membrane potential, increase in caspase-3 activity and lysosomal membrane leakiness before cell lysis ensued. All of the above mentioned cisplatin-induced oxidative stress cytotoxicity markers were significantly (p?<?0.05) prevented by ROS scavengers, antioxidants, mitochondrial permeability transition (MPT) pore sealing agents, endocytosis inhibitors and adenosine triphosphate (ATP) generators. Our results also showed that CYP2E1 involves in cisplatin oxidative stress cytotoxicity mechanism and intracellular nitric oxide enhancement protects the RPT cells against the cisplatin-induced cytotoxicity.

3. It seems that cisplatin nephrotoxicity is associated with mutual mitochondrial/lysosomal potentiation (cross-talk) of oxidative stress in RPT cells. This cross-talk finally results in release of lysosomal digestive proteases and phospholipases and mitochondrial MPT pore opening leading to cytochrome c release and activation of caspases cascade which signal apoptosis.

     

Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria

The clinical use of cisplatin (cis-diamminedichloroplatinum II) is highly limited by its nephrotoxicity. The precise mechanisms involved in cisplatin-induced mitochondrial dysfunction in kidney have not been completely clarified. Therefore, we investigated in vivo the effects of cisplatin on mitochondrial bioenergetics, redox state, and oxidative stress as well as the occurrence of cell death by apoptosis in cisplatin-treated rat kidney. Adult male Wistar rats weighing 200–220 g were divided into two groups. The control group (n = 8) was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml per 100 g body weight), and the cisplatin group (n = 8) was given a single injection of cisplatin (10 mg/kg body weight, i.p.). Animals were sacrificed 72 h after the treatment. The cisplatin group presented acute renal failure characterized by increased plasmatic creatinine and urea levels. Mitochondrial dysfunction was evidenced by the decline in membrane electrochemical potential and the substantial decrease in mitochondrial calcium uptake. The mitochondrial antioxidant defense system was depleted, as shown by decreased GSH and NADPH levels, GSH/GSSG ratio, and increased GSSG level. Moreover, cisplatin induced oxidative damage to mitochondrial lipids, including cardiolipin, and oxidation of mitocondrial proteins, as demonstrated by the significant decrease of sulfhydryl protein concentrations and increased levels of carbonylated proteins. Additionally, aconitase activity, which is essential for mitochondrial function, was also found to be lower in the cisplatin group. Renal cell death via apoptosis was evidenced by the increased caspase-3 activity. Results show the central role of mitochondria and the intensification of apoptosis in cisplatin-induced acute renal failure, highlighting a number of steps that might be targeted to minimize cisplatin-induced nephrotoxicity.     

The cisplatin-irradiation combination suggests that apoptosis is not a major determinant of clonogenic death

It is commonly believed that tumor cells treated with anticancer agents, chemotherapy and/or radiation, die by apoptosis and that tumors which do not undergo apoptosis are resistant to treatment. In this study, we investigated the molecular basis underlying cisplatin cytotoxicity in the murine teratocarcinoma F9 cell line to see whether irradiation enhances cisplatin-induced cytotoxicity. We compared the apoptosis induced by chemo and/or radiotherapy with other cellular effects such as cell survival, clonogenic capability, cell cycle perturbation, expression of p53 and p53-related mRNAs, and necrosis. When combined with radiation, a clear additive cytotoxic effect of cisplatin was demonstrated. We found that both cisplatin and radiation induced cell death, but the level of induced apoptosis was low and there was no correlation with the results of the clonogenic assays: we noted a difference between cytotoxic effects in the clonogenic assay and the extent of apoptosis by fluorescence-activated cell sorter analysis, suggesting that cell killing reflected not only apoptosis but also cell cycle arrest, and that apoptosis, cell kinetics and clonogenicity suppression were independent processes.     

Maitake β-glucan enhances therapeutic effect and reduces myelosupression and nephrotoxicity of cisplatin in mice

Cisplatin is broadly used clinically as an anticancer drug. Despite its significant anticancer activity, cisplatin-induced nephrotoxicity and myelosuppression limit its use. MD-Fraction is glucan purified from maitake (Grifola frondosa), which has β-1, 6-main chain with β-1, 3-branches, has been reported to exhibit antitumor and antimetastatic activities by enhancing the immune system. In this study, we demonstrate that MD-Fraction in combination with cisplatin significantly enhanced antitumor and antimetastatic activity compared to cisplatin alone. MD-Fraction reduced decreases in body weight, spleen weight and the number of immunocompetent cells such as macrophages, DCs and NK cells in cisplatin-treated mice. MD-Fraction also induced IL-12p70 production by splenocytes, resulting in increased NK cell activity in cisplatin-treated mice. MD-Fraction significantly increased the mRNA expression of GM-CSF, G-CSF, M-CSF, IFN-γ, IL-12 p40 in splenocytes and reduced the decrease in the number of CFU-GM colonies in cisplatin-treated bone marrow. These facts suggest that MD-Fraction can reduce cisplatin-induced myelosuppression. Moreover, treatment with MD-Fraction significantly reduced cisplatin-induced nephrotoxicity accompanied by increases in serum creatinine level, necrosis and apoptosis of renal tubular cells. These results suggest that MD-Fraction in combination with cisplatin cannot only enhance antitumor and antimentastatic acitivity, but also reduce cisplatin-induced myelotoxicity and nephrotoxicity.