Cardiotoxin III inhibits proliferation and migration of oral cancer cells through MAPK and MMP signaling.

Cardiotoxin III (CTXIII), isolated from the snake venom of Formosan cobra Naja naja atra, has previously been found to induce apoptosis in many types of cancer. Early metastasis is typical for the progression of oral cancer. To modulate the cell migration behavior of oral cancer is one of the oral cancer therapies. In this study, the possible modulating effect of CTXIII on oral cancer migration is addressed. In the example of oral squamous carcinoma Ca9-22 cells, the cell viability was decreased by CTXIII treatment in a dose-responsive manner. In wound-healing assay, the cell migration of Ca9-22 cells was attenuated by CTXIII in a dose- and time-responsive manner. After CTXIII treatment, the MMP-2 and MMP-9 protein expressions were downregulated, and the phosphorylation of JNK and p38-MAPK was increased independent of ERK phosphorylation. In conclusion, CTXIII has antiproliferative and -migrating effects on oral cancer cells involving the p38-MAPK and MMP-2/-9 pathways.

Satellite cells produce neural chemorepellent semaphorin 3A upon muscle injury.

Regenerative mechanisms that regulate intramuscular motor innervation. including configuration of the neuromuscular connections are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of satellite cells as a key source of a secreted neural chemorepellent semaphorin 3A (Sema3A) expression. In order to verify this concept, there is still a critical need to provide direct evidence to show the up-regulation of Sema3A protein in satellite cells in vivo upon muscle injury. The present study employed a Sema3A/MyoD double-immunohistochemical staining for cryo-sections prepared from cardiotoxin injected gastrocnemius muscle of adult mouse lower hind-limb. Results clearly demonstrated that Sema3A expression was up-regulated in myogenic differentiation-positive satellite cells at 4-12 days post-injury period, the time that corresponds to the cell differentiation phase characterized by increasing myogenin messenger RNA expression. This direct proof encourages a possible implication of satellite cells in the spatiotemporal regulation of extracellular Sema3A concentrations, which potentially ensures coordinating a delay in neurite sprouting and re-attachment of motoneuron terminals onto damaged muscle fibers early in muscle regeneration in synchrony with recovery of muscle-fiber integrity.

Prostatic involution after intraprostatic injection of cobra toxin.

PURPOSE: We evaluated the comparative effects of intraprostatic injection of cobra cardiotoxin D and botulinum toxin type A on prostate structure in the rat model. MATERIALS AND METHODS: A total of 18 Sprague-Dawley® rats weighing 500 to 600 gm received a single 0.1 ml injection of saline (6), botulinum toxin type A (6) or the cardiotoxin D (6) component of cobra (Naja naja atra) toxin in the right and left ventral lobes of the prostate. At 14 days the rats were sacrificed. The prostate glands were harvested, weighed and processed for immunohistochemical and morphological studies. RESULTS: Prostate glands injected with cardiotoxin D showed significantly decreased weight compared to that of prostates injected with botulinum toxin type A and the saline control. Prostatic atrophy in the glandular component with flattening of the epithelial lining was seen histologically in rats that received botulinum toxin and cardiotoxin D. Each group injected with cardiotoxin D and botulinum toxin showed a significant increase in the number of apoptotic cells compared with controls while only the botulinum toxin group showed a significant increase in the number of proliferating cells. Only rats injected with botulinum toxin had body weight loss. CONCLUSIONS: Our study shows that intraprostatic injection of cobra cardiotoxin D induces prostatic atrophy and leads to a decrease in prostatic weight greater than that of intraprostatic injection of botulinum toxin type A. No systemic effects, such as decreased body weight, were noted after cardiotoxin D injection. Further studies are warranted but the statistically significant decrease in the number of proliferating cells implies a prolonged effect of cardiotoxin D.

Vaccine site inflammation potentiates idiotype DNA vaccine-induced therapeutic T cell-, and not B cell-, dependent antilymphoma immunity.

Lymphoma idiotype protein vaccines have shown therapeutic potential in previous clinical studies, and results from a completed pivotal, phase 3 controlled trial are promising. However, streamlined production of these patient-specific vaccines is required for eventual clinical application. Here, we show that second-generation, chemokine-fused idiotype DNA vaccines, when combined with myotoxins that induced sterile inflammation with recruitment of antigen-presenting cells at vaccination sites, were exceptional in their ability to provoke memory antitumor immunity in mice, compared with several TLR agonists. The combined vaccination strategy elicited both antigen-specific T-cell responses and humoral immunity. Unexpectedly, vaccine-induced tumor protection was intact in B cell-deficient mice but was abrogated completely by T-cell depletion in vivo, suggesting T-cell dependence. Furthermore, the optimal effect of myotoxins was observed with fusion vaccines that specifically targeted antigen delivery to antigen-presenting cells and not with vaccines lacking a targeting moiety, suggesting that the rational vaccine design will require combination strategies with novel, proinflammatory agents and highly optimized molecular vaccine constructs. These studies also challenge the paradigm that antibody responses are the primary of idiotype-specific antitumor effects and support the optimization of idiotype vaccines designed to induce primarily T-cell immunity.

Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype.

OBJECTIVE: Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. METHODS: In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin(-/+), c-kit(-/+), Sca-1(-/+)) from GFP(+) (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). RESULTS: The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP(+)/CD45(-)) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP(+)/CD45(-) cells were also cytokeratin(+). Mobilization further increased GFP(+)/CD45(-) cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP(+)/CD45(-) in radiation-injured mice transplanted with Lin(-), c-kit(+), or Sca-1(+) marrow cells. Lin(+), c-kit(-), and Sca-1(-) subpopulations did not significantly engraft the lung. CONCLUSIONS: We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell-producing marrow cell has the phenotype of a hematopoietic stem cell.

Individual analysis of mice vaccinated against a weakly immunogenic self tumor-specific antigen reveals a correlation between CD8 T cell response and antitumor efficacy.

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.

Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin.

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.

Highly coordinated gene regulation in mouse skeletal muscle regeneration.

Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection. Among 16,267 gene elements surveyed, 3,532 elements showed at least a 2.5-fold change at one or more time points during a 14-day time course. Hierarchical cluster analysis and semiquantitative reverse transcription-PCR showed induction of genes important for cell cycle control and DNA replication during the early phase of muscle regeneration. Subsequently, genes for myogenic regulatory factors, a group of imprinted genes and genes with functions to inhibit cell cycle progression and promote myogenic differentiation, were induced when myogenic stem cells started to differentiate. Induction of a majority of these genes, including E2f1 and E2f2, was abolished in muscles lacking satellite cell activity after gamma radiation. Regeneration was severely compromised in E2f1 null mice but not affected in E2f2 null mice. This study identifies novel genes potentially important for muscle regeneration and reveals highly coordinated myogenic cell proliferation and differentiation programs in adult skeletal muscle regeneration in vivo.

Alpha1-syntrophin-deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration.

Alpha1-syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of alpha1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of alpha1-syntrophin-null (alpha1syn-/-) mice. After the treatment, alpha1syn-/- muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, alpha1syn-/- mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in alpha1syn-/- regenerating muscles. Our results suggest that the lack of alpha1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients.

30-day intravenous administration of VRCTC-310-ONCO in rabbits.

VRCTC-310-ONCO, an agent based on the snake phospholipase A2 (crotoxin), is currently under clinical development. After phase I study in patients by intramuscular administration, the interest of intravenous (IV) dosing arose. To evaluate IV administration of VRCTC-310-ONCO in rabbits, ten animals were subjected to surgical implant of fixed jugular catheter, by which they received daily IV doses of 0.03 mg/kg body weight of VRCTC-310-ONCO for 30 days (n = 8) or saline (n = 2). The procedure was well tolerated in all rabbits. One of the animals died after the sixth dose of VRCTC-310-ONCO with CNS involvement; two additional rabbits required dose-reduction. All other rabbits achieved 30 days of treatment and were sacrificed. All rabbits (even controls) developed lymphocytosis and mild anaemia, without changes in blood neutrophils. No changes were found in serum transaminases (GOT and GPT), cholesterol, triglycerides, and y-glutamyl transpeptidase. At necropsy, chronic granulation tissue was found surrounding the implant in all rabbits. VRCTC-3 10-ONCO-treated rabbits presented generalised and marked swelling of hepatocytes, with areas of cytoplasmic vacuolisation. No abnormalities were found in kidney, heart, lung, spleen, adrenal gland, uterus, testes and ovary. Additional studies with IV route for VRCTC-310-ONCO, including humans, are required to define its toxicity in the clinical setting.

In vivo effect of snake phospholipase A2 (crotoxin+cardiotoxin) on serum IL-1alpha, TNF-alpha and IL-1ra level in humans.

VRCTC-310-Onco (crotoxin, a secretory phospholipase A2+cardiotoxin) is under development as an anti-neoplastic agent. Pro-inflammatory cytokines TNF-alpha and interleukin 1 alpha (IL-1alpha) and anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) were measured with commercial ELISA kits in sera corresponding to 23 cycles with doses between 0.0025 and 0.023 microg/kg body weight, obtained during the phase I trial of VRCTC-310-Onco. Neither serum TNF-alpha nor IL-1alpha did change significantly after VRCTC-310-Onco. Basal IL-1ra was 794 +/- 97 pg/ml, by 3 h it was similar, 651 +/- 99 pg/ml and at 24 h p.i. it increased to 1197 +/- 122 pg/ml (P<0.001). The increase was dose-dependent. The addition of dexamethasone (required to reduce pain with the highest doses) inhibited IL-1alpha and enhanced the induction of IL-1ra by VRCTC-310-Onco. Summing up, in vivo, in humans, in the dose range tested, VRCTC-310-Onco induces IL-1ra, and does not consistently modify IL-1alpha or TNF-alpha serum levels.

One-month safety study of intraperitoneal VRCTC-310-Onco (crotoxin + cardiotoxin) in rats.

To evaluate the toxicity of VRCTC-310-Onco (Crotalus durissus terrificus crotoxin + cardiotoxin from Naja naja atra), 10 Sprague-Dawley rats were implanted with intraperitoneal slow-release devices and subjected to treatment with 0.5 microgram/g body weight/d for 14 days. Biochemical evidence at days 7 and 14 showed blood, muscular, renal and metabolic disturbance, mostly reversed by day 28. No significant changes were found in necropsy. The limited toxicity of i.p. VRCTC-310-Onco in rats deserves further study.

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.

Tumor regression of advanced carcinomas following intra- and/or peri-tumoral inoculation with VRCTC-310 in humans: preliminary report of two cases.

The authors report their clinical experience with VRCTC-310 in two patients suffering with advanced cancer in which the skin was severely compromised. VRCTC-310 is a combination of the snake venoms crotoxin (CT) and cardiotoxin (CD). The local (peritumoral) treatment with the drug (0.O14 mg/kg/week during 6 weeks) provoked the complete disappearance of a relapsed skin squamous cell cancer in one patient. The other patient was an aged woman with local-advanced breast cancer (carcinoma en cuirasse) who was inoculated intra-and-peritumoral with VRCTC-310. After 6 weekly courses (0.014 mg/kg/week) with the drug a > 80% tumor reduction was seen. A 133 days follow-up demonstrated not only an objective complete response of the primary tumor mass, but the disappearance of supraclavicular tumor mass as well a significant reduction in lymphangitis. To our knowledge, this is the first communication about the in vivo antitumoral activity of VRCTC-310 when injected locally to humans. Further studies are now in progress.

Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle.

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.

Toxicity of the novel animal-derived anticancer agent, VRCTC-310: acute and subchronic studies in beagle dogs.

Acute and subchronic toxicities of VRCTC-310, a combination product of crotoxin (CT) and cardiotoxin (CD), which has shown antitumor activity in vivo, have been studied in Beagle dogs. Single i.m. doses of 0.25, 0.5 and 1.0 mg/kg resulted in dose-dependent local muscular toxicity consisting of myofiber atrophy, interstitial edema and macrophage infiltration. Also, AST, ALT and LDH levels increased on day 2, returning to normal values on days 6-8. Local lesions were absent after recovery on day 45. At 2.0 mg/kg, signs of neurotoxicity (ataxia) appeared, in addition to vomitus, salivation, hematuria and myotoxicity in tongue and diaphragm on day 8. Local lesions healed with fibrosis at the site of injection on day 45. Administration of fixed (0.025 and 0.05 mg/kg) or escalating (0.025-0.1 mg/kg) daily doses for 30 days also produced local muscular damage, which was absent at day 75. The increases in AST, ALT and LDH serum activities on days 2-4 were independent of dosing schedule and sharply decreased on day 8, despite continuation of treatment. An escalating dose schedule of 0.025-2.0 mg/kg showed local muscle damage at the site of injection on day 31, however, there were no lesions of myotoxicity in the tongue or diaphragm and no clinical signs of neurotoxicity were observed. Animals tolerated the subchronic treatment better than the acute. The resolution of serum enzymes to normal values during treatment may be attributed to a decrease of sensitivity to VRCTC-310-mediated myotoxic effects.

[Observations on cytolysis by cobra venom. Effect of sucrose loading]. / Osservazioni sulla citolisi da veleno di cobra. Effetto del carico con saccarosio.

LS cells cultivated for 4 days in sucrose-containing medium (Su) are more fragile than cells cultivated in medium devoid of this sugar (TN) when they are incubated with Naja-Naja venom; in fact the 51Cr radioactivity released into the medium by previously labelled cells is increased for the sucrose ones. Phospholipase does not seem to contribute to the lytic activity of the venom, which is dependent on the direct lytic factor: neither varying the temperature of incubation nor adding EDTA to the incubation medium modifies the results. Adding Ca++ decreases the 51Cr radioactivity released particularly by the cells grown in TN, suggesting a stabilizing action of this cation.