Oxaliplatin down-regulates survivin by p38 MAP kinase and proteasome in human colon cancer cells

Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. Survivin has been proposed as a cancer target, which highly expressed in most cancer cells but not normal adult cells. In this study, we investigated the regulation of survivin expression by exposure to oxaliplatin in human colon cancer cells. Oxaliplatin (3–9 μM for 24 h) markedly induced cytotoxicity, proliferation inhibition and apoptosis in the human RKO colon cancer cells. The survivin protein expression of RKO cells is dramatically reduced by oxaliplatin; however, the survivin gene expression is slightly altered. The survivin blockage of oxaliplatin elevated caspase-3 activation and apoptosis in RKO cells. Over-expression of survivin proteins by transfection with a survivin-expressed vector resisted the oxaliplatin-induced cancer cell death. Meantime, oxaliplatin elicited the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated oxaliplatin-induced cancer cell death. In addition, oxaliplatin increased the levels of phospho-p53 (Ser-15) and total p53 proteins. Inhibition of p53 expression by a specific p53 inhibitor pifithrin-α reduced the phosphorylated p38 MAP kinase and active caspase-3 proteins in the oxaliplatin-exposed RKO cells. In contrast, SB202190 did not alter the oxaliplatin-induced p53 protein level. Furthermore, treatment with a specific proteasome inhibitor MG132 restored survivin protein level in the oxaliplatin-treated colon cancer cells. Taken together, our results demonstrate for the first time that survivin is down-regulated by p38 MAP kinase and proteasome degradation pathway after treatment with oxaliplatin in the human colon cancer cells.     

Beta2-agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the beta2-adrenoceptor agonist (beta2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

Expression of Hsp27 correlated with rat detrusor contraction after acute urinary retention

Heat shock protein 27 (Hsp27) can regulate actin cytoskeleton dynamics and contractile protein activation. This study investigates whether Hsp27 expression is related to bladder contractile dysfunction after acute urinary retention (AUR). Female rats were randomized either to AUR by urethral ligation or to normal control group. Bladder and smooth muscle strip contraction at time points from 0 h to 7 days after AUR were estimated by cystometric and organ bath studies. Hsp27 expression in bladder tissue at each time point was detected with immunofluorescence, Western blots, and real-time PCR. Expression of the three phosphorylated forms of Hsp27 was detected by Western blots. Smooth muscle ultrastructure was observed by transmission electron microscopy. Data suggest that maximum detrusor pressure and both carbachol-induced and spontaneous detrusor strip contraction amplitude decreased gradually for the duration from 0 to 6 h, and then increased gradually to near-normal values at 24 h. Treatment of muscle strips with the p38MAK inhibitor, SB203580, inhibited carbachol-induced contractions. Smooth muscle ultrastructure damage was the highest at 6 h after AUR, and then lessened gradually during next 7 days, and ultrastructure was close to normal. Expressions of Hsp27 mRNA and protein and the proteins of the three phosphorylated forms were higher at 0 h, decreased to lower levels up to 6 h, and then gradually increased. Therefore, we conclude that rat bladder contractile function after AUR worsens during 0–6 h, and then gradually recovers. The findings of the current study suggest that Hsp27 modulates bladder smooth muscle contraction after AUR, and that phosphorylation of Hsp27 may be an important pathway modulating actin cytoskeleton dynamics in bladder smooth muscle contraction and reconstruction after injury.     

Characterization of control and immobilized skeletal muscle: an overview from genetic engineering.

To elucidate the molecular basis of muscle atrophy, we have performed the serial analysis of gene expression (SAGE) method with control and immobilized muscles of 10 rats. The genes that expressed >0.5% in muscle are involved in the following three functions: 1) contraction (troponin I, C and T; myosin light chain 1-3; actin; tropomyosin; and parvalbumin), 2) energy metabolism (cytochrome c oxidase I and III, creatine kinase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, ATPase 6, and aldolase A), and 3) housekeeping (lens epithelial protein). Muscle atrophy appears to be caused by changes in mRNA levels of specific regulators of proteolysis, protein synthesis, and contractile apparatus assembling, such as polyubiquitin, elongation factor 2, and nebulin. Immobilization has produced a decrease more than threefold in gene expression of enzymes involved in energy metabolism, especially ATPase, cytochrome c oxidase, NADH dehydrogenase, and protein phosphatase 1. Differential gene expressions of selenoprotein W and uroporphyrinogen decarboxylase, which can be involved in oxidative stress, were also observed. Other genes with various functions, such as cholesterol metabolism and growth factors, were also differentially expressed. Moreover, novel genes regulated by immobilization were discovered. Thus, the current study allows a better understanding of global muscle characteristics and the molecular mechanisms of sedentarity and sarcopenia.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.     

Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr²⁺ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr²⁺ concentration ([Sr²⁺]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr²⁺ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr²⁺ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (–log₁₀ [Sr²⁺]) curves is tightly (r² = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr²⁺ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr²⁺, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers. muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium     

Exenatide Facilitates Recovery from Oxaliplatin-Induced Peripheral Neuropathy in Rats

Background

Oxaliplatin has widely been used as a key drug in the treatment of colorectal cancer; however, it causes peripheral neuropathy. Exenatide, a glucagon-like peptide-1 (GLP-1) agonist, is an incretin mimetic secreted from ileal L cells, which is clinically used to treat type 2 diabetes mellitus. GLP-1 receptor agonists have been reported to exhibit neuroprotective effects on the central and peripheral nervous systems. In this study, we investigated the effects of exenatide on oxaliplatin-induced neuropathy in rats and cultured cells.

Methods

Oxaliplatin (4 mg/kg) was administered intravenously twice per week for 4 weeks, and mechanical allodynia was evaluated using the von Frey test in rats. Axonal degeneration was assessed by toluidine blue staining of sciatic nerves.

Results

Repeated administration of oxaliplatin caused mechanical allodynia from day 14 to 49. Although the co-administration of extended-release exenatide (100 μg/kg) could not inhibit the incidence of oxaliplatin-induced mechanical allodynia, it facilitated recovery from the oxaliplatin-induced neuropathy with reparation of axonal degeneration. Inhibition of neurite outgrowth was evaluated in cultured pheochromocytoma 12 (PC12) cells. Exenatide inhibited oxaliplatin-induced neurite degeneration, but did not affect oxaliplatin-induced cell injury in cultured PC12 cells. Additionally, extended-release exenatide had no effect on the anti-tumor activity of oxaliplatin in cultured murine colon adenocarcinoma 26 (C-26) cells or C-26 cell-implanted mice.

Conclusion

These results suggest that exenatide may be useful for treating peripheral neuropathy induced by oxaliplatin in colorectal cancer patients with type 2 diabetes.     

O-linked N-acetylglucosaminylation is involved in the Ca2+ activation properties of rat skeletal muscle

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction.     

Oxaliplatin-induced oxidative stress in nervous system-derived cellular models: Could it correlate with in vivo neuropathy?

Oxaliplatin is a platinum-organic drug with antineoplastic properties used for colorectal cancer. With respect to the other platinum derivates oxaliplatin induces only a mild hematological and gastrointestinal toxicity. Its limiting side effect is its neurotoxicity, which results in a sensory neuropathy. Repeated oxaliplatin treatment in the rat led to a neuropathic pain characterized by a significant oxidative damage throughout the nervous system. The natural antioxidants silibinin and α-tocopherol reduce redox alteration and prevent pain. Starting from the “oxidative hypothesis” as a molecular basis of chemotherapy-induced neurotoxicity, we decided to explore deep inside the mechanisms of oxaliplatin neurotoxicity and search for a cellular system useful for screening antioxidant compounds that can reduce oxaliplatin neurotoxicity. Focusing on various constituents of the central nervous system, we used the neuronal-derived cell line SH-SY5Y and primary cultures of rat cortical astrocytes. Oxaliplatin significantly increased superoxide anion production and induced lipid peroxidation (malonyldialdehyde levels) and protein (carbonylated proteins) and DNA oxidation (8-OH-dG levels). Silibinin and α-tocopherol (10 µM) were able to reduce the oxidative damage in both cell types. These antioxidants fully protected astrocytes from the caspase 3 apoptotic signaling activation induced by oxaliplatin. The damage prevention effects of silibinin and α-tocopherol on nervous system-derived cells did not interfere with the oxaliplatin antineoplastic in vitro mechanism as evaluated on a human colon adenocarcinoma cell line (HT29). Moreover, neither silibinin nor α-tocopherol modified the oxaliplatin-induced apoptosis in HT29 cells, suggesting a different antiapoptotic profile in normal vs tumoral cells for these antioxidant compounds. In conclusion, because data obtained in in vitro cellular models parallel the in vivo study we propose cell models to investigate oxaliplatin neurotoxicity and to screen possible therapeutic adjuvant agents.     

Aging and neural control of the GI tract: V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins

The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Diaphragm muscle fatigue resistance during postnatal development.

Changes in the contractile and fatigue properties of the cat diaphragm muscle were examined during the first 6 wk of postnatal development. Both twitch contraction time and half-relaxation time decreased progressively with age. Correspondingly, the force-frequency curve was shifted to the left early in development compared with adults. The ratio of peak twitch force to maximum tetanic force decreased with age. Fatigue resistance of the diaphragm was highest at birth and then progressively decreased with age. At birth, most diaphragm muscle fibers stained darkly for myofibrillar adenosinetriphosphatase after alkaline preincubation and thus would be classified histochemically as type II. During subsequent postnatal development, the proportion of type I fibers (lightly stained for adenosinetriphosphatase) increased while the number of type II fibers declined. At birth, type I fibers were larger than type II fibers. The size of both fiber types increased with age, but the increase in cross-sectional area was greater for type II fibers. On the basis of fiber type proportions and mean cross-sectional areas, type I fibers contributed 15% of total muscle mass at birth and 25% in adults. Thus postnatal changes in diaphragm contractile and fatigue properties cannot be attributed to changes in the relative contribution of histochemically classified type I and II fibers. However, the possibility that these developmental changes in diaphragm contractile and fatigue properties correlated with the varying contractile protein composition of muscle fibers was discussed.     

Adaptation to low-protein diet increases inhibition of gastric emptying by CCK

Chronic nutritional disorders such as protein malnutrition are associated with delayed gastric emptying and increased postprandial cholecystokinin (CCK) levels. This study investigated the mechanisms involved in gastric emptying adaptation to low-protein diet. Two groups of 12 rats were adapted to a low-protein (LPD) or standard diet (SD) for 3 weeks. As compared to rats fed a SD, in rats adapted to a LPD gastric emptying was delayed, whereas postprandial CCK levels were increased. LPD enhanced antral muscle contractile response to CCK and cerulein without altering response to acetylcholine. This increased contractility was associated with up-regulation of CCK-A receptor mRNA levels in antral muscle. Our data suggest that modulation of gastric emptying after adaptation to a low-protein diet involves up-regulation of both CCK-A receptors and CCK-induced contraction of antral smooth muscle.     

Effects of nicorandil on isolated smooth muscle cells from guinea-pig taenia caeci

1. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles. 2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells. 3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile proteins in an indirect manner in guinea-pig taenia caeci.     

Autophagy Protects against Oxaliplatin-Induced Cell Death via ER Stress and ROS in Caco-2 Cells

Oxaliplatin is included in a number of effective combination regimens used as first and subsequent lines of therapy for metastatic colorectal cancer. Accumulating evidence indicates that autophagy plays a significant role in response to cancer therapy. However, the role of autophagy in oxaliplatin-induced cell death remains to be clarified. In this study, we showed that oxaliplatin induced cell death and autophagy in Caco-2 colorectal cancer cells. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or Beclin1) enhanced the cell death and reactive oxygen species (ROS) production induced by oxaliplatin in Caco-2 cells. Blocking oxaliplatin-induced ROS production by using ROS scavengers (NAC or Tiron) decreased autophagy. Furthermore, numerous dilated endoplasmic reticula (ER) were present in oxaliplatin-treated Caco-2 cells, and blocking ER stress by RNA interference against candidate of metastasis-1 (P8) and C/EBP-homologous protein (CHOP) decreased autophagy and ROS production. Taken together, these data indicate that oxaliplatin activates autophagy as a cytoprotective response via ER stress and ROS in human colorectal cancer cells.