Resistance to 5-Fluorouracil chemotherapy is a major cause of therapeutic failure in colon cancer... more Resistance to 5-Fluorouracil chemotherapy is a major cause of therapeutic failure in colon cancer cure. Development of combined therapies constitutes an effective strategy to inhibit cancer cells and prevent the emergence of drug resistance. For this purpose, we investigated the anti-tumoral effect of thirteen phenolic compounds, from the Tunisian quince Cydonia oblonga Miller, alone or combined to 5-FU, on the human 5-FU-resistant LS174-R colon cancer cells in comparison to parental cells. Our results showed that only Kaempferol was able to chemo-sensitize 5-FU-resistant LS174-R cells. This phenolic compound combined with 5-FU exerted synergistic inhibitory effect on cell viability. This combination enhanced the apoptosis and induced cell cycle arrest of both chemo-resistant and sensitive cells through impacting the expression levels of different cellular effectors. Kaempferol also blocked the production of reactive oxygen species (ROS) and modulated the expression of JAK/STAT3, MA...
The Vascular Endothelial Growth Factor "VEGF" plays a pivotal role in the stimulation o... more The Vascular Endothelial Growth Factor "VEGF" plays a pivotal role in the stimulation of angiogenesis. The VEGF isoforms (A-D) and PlGF act in a coordinate fashion to develop the vascular network. Numerous proteins closely related in structure and function to VEGF-A have been reported and were grouped in the VEGF family. Some predators make use of VEGF-like molecules with devastating results for their prey. VEGF-E, investigated in 1994, is encoded by the parapoxvirus (Orf virus). VEGF-F is a common term designating molecules which were isolated from snake venom (also known as svVEGF). These proteins are disulphide-linked homodimers of 110 amino acids each and have a molecular weight of approximately 25 kDa. Their primary structures show approximately 50% identity to VEGF-A. However, unlike VEGF-A, they do not contain any N-linked glycosylation sites. They interact with heparin but have a different binding domain from that of VEGF-A. Among species, these svVEGFs vary extens...
Additional file: Figure S1. The effect of combination of Peph phenolic compounds on proliferation... more Additional file: Figure S1. The effect of combination of Peph phenolic compounds on proliferation of LS174 cells compared to total Peph extract. Human colon adenocarcinoma LS174 cells were treated for 72Â h with different combinations (A. 3 combinations, B. 4 combinations, C. 5 combinations) of Peph phenolic compounds. All compounds where tested at equivalent concentrations to that present in 5Â Îźg/ml of the total peel polyphenolic extract. Cell viability was determined by MTT assay.
The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been ... more The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been correlated with the overexpression of the mesenchymal-epithelial transition factor (c-Met) receptor. Thus, the hepatocyte growth factor (HGF)/c-Met signaling axis has gained considerable attention as a valid molecular target for breast cancer therapy. This study reports for the first time the discovery of the 13 -oxophorbines pheophorbide A and protopheophorbide A along with chlorophyllide A from Ziziphus lotus, an edible typical Tunisian plant, as the potent antiproliferative compounds against the human breast cancer cells MDA-MB-231 and MCF-7. Compared to other compounds, protopheophorbide A exerted the highest light-independent antiproliferative effect against the metastatic TNBC MDA-MB-231 cells (IC = 6.5 μM). In silico, this compound targeted the kinase domain of multiple c-Met crystal structures. It potently inhibited the kinase domain phosphorylation of wild and mutant c-Met in Z...
The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search f... more The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced ...
Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has eme... more Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK1/2, Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of F...
Evidence-Based Complementary and Alternative Medicine, 2016
We report the chemical composition and anti-Leishmaniaand antioxidant activity ofArtemisia campes... more We report the chemical composition and anti-Leishmaniaand antioxidant activity ofArtemisia campestrisL. andArtemisia herba-albaAsso. essential oils (EOs). Our results showed that these extracts exhibit different antioxidant activities according to the used assay. The radical scavenging effects determined by DPPH assay were of IC50= 3.3 mg/mL and IC50= 9.1 mg/mL forArtemisia campestrisandArtemisia herba-albaessential oils, respectively. However, antioxidant effects of both essential oils, determined by ferric-reducing antioxidant power (FRAP) assay, were in the same range (2.3 and 2.97 mg eq EDTA/g EO, resp.), while theArtemisia herba-albaessential oil showed highest chelating activity of Fe2+ions (27.48 mM Fe2+). Interestingly, we showed that both EOs possess dose-dependent activity againstLeishmania infantumpromastigotes with IC50values of 68 μg/mL and 44 μg/mL forA. herba-albaandA. campestris, respectively. We reported, for the first time, that antileishmanial activity of both EOs...
Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterize... more Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associ...
cells through a unique mechanism that involves a calcium-dependent translocation of its catalytic... more cells through a unique mechanism that involves a calcium-dependent translocation of its catalytic domain across the plasma membrane (Karst et al., 2012; Veneziano et al., 2013). This mechanism of entry is still poorly understood and the main goal of our project is to unravel this remarkable process. We have recently obtained a functional refolded monomeric state of the toxin. Our studies demonstrate that post-translational acylation and calcium binding are critical for CyaA folding into a stable monomeric and active form.
BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tuneta... more BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.
Background: Development of alternative cancer-specific drugs would be of paramount importance to ... more Background: Development of alternative cancer-specific drugs would be of paramount importance to overcome toxicity toward normal tissues and tumor resistance. Here, we investigated the potential anti-tumoral effect of peel (Peph) and pulp polyphenolic extracts from the Tunisian quince Cydonia oblonga Miller on both no-tumorigenic cells NIH 3T3 Fibroblasts and HEK 293 cells and human colon adenocarcinoma LS174 cells. Methods: Cell proliferation and cytotoxicity were measured with MTT and LDH assays respectively. Cell cycle distribution and the apoptosis levels were assessed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were determined using the fluorescent probe CM-H2DCFDA. Western blot was used to further characterize cell death and analyze the signaling pathways affected by Peph treatment. The expression level of VEGF-A was evaluated by real time quantitative PCR and further verified by quantifying the secreted cytokines by enzyme-linked immunosorbent assay. We found that Peph extract displayed the highest anti-proliferative effect specifically on LS174 cells. However, each Peph phenolic compound alone did not exhibit any anti-proliferative activity, suggesting a synergistic effect of phenolic molecules. Such effect was associated with a cell cycle arrest in the G1/S phase, a caspase-independent apoptosis and an increase of the ROS production. Peph extract inhibited the pro-survival signaling pathway NFκB and suppressed the expression of various cellular markers known to be involved in cell cycling (cyclin D1) and angiogenesis (Vascular Endothelial Growth Factor, VEGF). Interestingly, the combination Peph extract and 5-FU exerted synergistic inhibitory effect on cell viability. These data propose the quince Peph extract as a promising cost effective non toxic drug to employ alone or in combination with conventional anti-colorectal cancer. Moreover, quince rich regimen may prevent the development and the progress of colon cancer.
BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium c... more BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhαIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect α-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8–10, 19–22, and 38–43. To evaluate their functional role, three BotXIV/LqhαIT chimeras were designed by transferring the corresponding LqhαIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular α/β fold. Interestingly, substitution of residues 8–10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of α-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure–function relationships between insect α-toxins and α-like scorpion toxins are probably more complex than expected.
Toxicon : official journal of the International Society on Toxinology, 1997
One contractive and two depressant toxins active on insect were purified by high-performance liqu... more One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = ...
We purified to homogeneity from venom of Buthus occitanus tunetanus seven toxins active on the co... more We purified to homogeneity from venom of Buthus occitanus tunetanus seven toxins active on the cockroach Periplaneta germanica. These toxins belong to previously defined groups, contracturant and depressive relaxant. A dose of 50-150 ng of toxins is able to kill 50% of injected P. germanica but only the contracturant toxins show toxicity to C57B16 mice injected i.c.v, with a DLs0 of 1 #g. The binding of 125I AaHIT (an excitatory contracturant toxin) to P. americana synaptosomes is inhibited at the same level as AaHIT, by the following depressive toxins: BotlT4, BotlT5 and BotlT6 and also by a contracturant toxin BotlT2. The half-effect is about 1-4 nM. 10 -6 M of delayed contracturant toxins BotlTl, BotIT3 and BotlT7 did not inhibit the binding of t25I AaHIT to its receptor. BotlT1, BotlT2, BotlT3 and BotlT7 cross-react with AaHIT as observed in ELISA tests using anti-AaHIT antibodies. These toxins are not recognized by anti-LqqlT2 antibodies. BotlT4 and BotlT5, which differ by a unique substitution in position 6, Lys instead of Arg, show about 80% sequence homology with LqqlT2. In contrast, BotlT4, BotlT5 and BotlT6 are recognized exclusively by anti-LqqlT2 antibodies. Purified Bot insect toxins, when applied on the isolated giant axon of the cockroach P. americana, show various effects. BotlT4, BotlT5 and BotlT6 are able to block the action potential and progressively suppress the Na ÷ current. BotlT1, BotlT3 and BotlT7 prolong the inactivation phase of the action potential, resulting in a large augmentation of Na ÷ current. BotlT2 induces limited depolarization concomitantly with the development of depolarization after potential, repetitive activity and later plateau potentials terminated by bursts.
A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Per... more A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetunus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and a-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As a-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike a-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na' channels by decreasing the peak Na+current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na+-channel activation.
Resistance to 5-Fluorouracil chemotherapy is a major cause of therapeutic failure in colon cancer... more Resistance to 5-Fluorouracil chemotherapy is a major cause of therapeutic failure in colon cancer cure. Development of combined therapies constitutes an effective strategy to inhibit cancer cells and prevent the emergence of drug resistance. For this purpose, we investigated the anti-tumoral effect of thirteen phenolic compounds, from the Tunisian quince Cydonia oblonga Miller, alone or combined to 5-FU, on the human 5-FU-resistant LS174-R colon cancer cells in comparison to parental cells. Our results showed that only Kaempferol was able to chemo-sensitize 5-FU-resistant LS174-R cells. This phenolic compound combined with 5-FU exerted synergistic inhibitory effect on cell viability. This combination enhanced the apoptosis and induced cell cycle arrest of both chemo-resistant and sensitive cells through impacting the expression levels of different cellular effectors. Kaempferol also blocked the production of reactive oxygen species (ROS) and modulated the expression of JAK/STAT3, MA...
The Vascular Endothelial Growth Factor "VEGF" plays a pivotal role in the stimulation o... more The Vascular Endothelial Growth Factor "VEGF" plays a pivotal role in the stimulation of angiogenesis. The VEGF isoforms (A-D) and PlGF act in a coordinate fashion to develop the vascular network. Numerous proteins closely related in structure and function to VEGF-A have been reported and were grouped in the VEGF family. Some predators make use of VEGF-like molecules with devastating results for their prey. VEGF-E, investigated in 1994, is encoded by the parapoxvirus (Orf virus). VEGF-F is a common term designating molecules which were isolated from snake venom (also known as svVEGF). These proteins are disulphide-linked homodimers of 110 amino acids each and have a molecular weight of approximately 25 kDa. Their primary structures show approximately 50% identity to VEGF-A. However, unlike VEGF-A, they do not contain any N-linked glycosylation sites. They interact with heparin but have a different binding domain from that of VEGF-A. Among species, these svVEGFs vary extens...
Additional file: Figure S1. The effect of combination of Peph phenolic compounds on proliferation... more Additional file: Figure S1. The effect of combination of Peph phenolic compounds on proliferation of LS174 cells compared to total Peph extract. Human colon adenocarcinoma LS174 cells were treated for 72Â h with different combinations (A. 3 combinations, B. 4 combinations, C. 5 combinations) of Peph phenolic compounds. All compounds where tested at equivalent concentrations to that present in 5Â Îźg/ml of the total peel polyphenolic extract. Cell viability was determined by MTT assay.
The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been ... more The failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been correlated with the overexpression of the mesenchymal-epithelial transition factor (c-Met) receptor. Thus, the hepatocyte growth factor (HGF)/c-Met signaling axis has gained considerable attention as a valid molecular target for breast cancer therapy. This study reports for the first time the discovery of the 13 -oxophorbines pheophorbide A and protopheophorbide A along with chlorophyllide A from Ziziphus lotus, an edible typical Tunisian plant, as the potent antiproliferative compounds against the human breast cancer cells MDA-MB-231 and MCF-7. Compared to other compounds, protopheophorbide A exerted the highest light-independent antiproliferative effect against the metastatic TNBC MDA-MB-231 cells (IC = 6.5 μM). In silico, this compound targeted the kinase domain of multiple c-Met crystal structures. It potently inhibited the kinase domain phosphorylation of wild and mutant c-Met in Z...
The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search f... more The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced ...
Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has eme... more Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK1/2, Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of F...
Evidence-Based Complementary and Alternative Medicine, 2016
We report the chemical composition and anti-Leishmaniaand antioxidant activity ofArtemisia campes... more We report the chemical composition and anti-Leishmaniaand antioxidant activity ofArtemisia campestrisL. andArtemisia herba-albaAsso. essential oils (EOs). Our results showed that these extracts exhibit different antioxidant activities according to the used assay. The radical scavenging effects determined by DPPH assay were of IC50= 3.3 mg/mL and IC50= 9.1 mg/mL forArtemisia campestrisandArtemisia herba-albaessential oils, respectively. However, antioxidant effects of both essential oils, determined by ferric-reducing antioxidant power (FRAP) assay, were in the same range (2.3 and 2.97 mg eq EDTA/g EO, resp.), while theArtemisia herba-albaessential oil showed highest chelating activity of Fe2+ions (27.48 mM Fe2+). Interestingly, we showed that both EOs possess dose-dependent activity againstLeishmania infantumpromastigotes with IC50values of 68 μg/mL and 44 μg/mL forA. herba-albaandA. campestris, respectively. We reported, for the first time, that antileishmanial activity of both EOs...
Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterize... more Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associ...
cells through a unique mechanism that involves a calcium-dependent translocation of its catalytic... more cells through a unique mechanism that involves a calcium-dependent translocation of its catalytic domain across the plasma membrane (Karst et al., 2012; Veneziano et al., 2013). This mechanism of entry is still poorly understood and the main goal of our project is to unravel this remarkable process. We have recently obtained a functional refolded monomeric state of the toxin. Our studies demonstrate that post-translational acylation and calcium binding are critical for CyaA folding into a stable monomeric and active form.
BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tuneta... more BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.
Background: Development of alternative cancer-specific drugs would be of paramount importance to ... more Background: Development of alternative cancer-specific drugs would be of paramount importance to overcome toxicity toward normal tissues and tumor resistance. Here, we investigated the potential anti-tumoral effect of peel (Peph) and pulp polyphenolic extracts from the Tunisian quince Cydonia oblonga Miller on both no-tumorigenic cells NIH 3T3 Fibroblasts and HEK 293 cells and human colon adenocarcinoma LS174 cells. Methods: Cell proliferation and cytotoxicity were measured with MTT and LDH assays respectively. Cell cycle distribution and the apoptosis levels were assessed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were determined using the fluorescent probe CM-H2DCFDA. Western blot was used to further characterize cell death and analyze the signaling pathways affected by Peph treatment. The expression level of VEGF-A was evaluated by real time quantitative PCR and further verified by quantifying the secreted cytokines by enzyme-linked immunosorbent assay. We found that Peph extract displayed the highest anti-proliferative effect specifically on LS174 cells. However, each Peph phenolic compound alone did not exhibit any anti-proliferative activity, suggesting a synergistic effect of phenolic molecules. Such effect was associated with a cell cycle arrest in the G1/S phase, a caspase-independent apoptosis and an increase of the ROS production. Peph extract inhibited the pro-survival signaling pathway NFκB and suppressed the expression of various cellular markers known to be involved in cell cycling (cyclin D1) and angiogenesis (Vascular Endothelial Growth Factor, VEGF). Interestingly, the combination Peph extract and 5-FU exerted synergistic inhibitory effect on cell viability. These data propose the quince Peph extract as a promising cost effective non toxic drug to employ alone or in combination with conventional anti-colorectal cancer. Moreover, quince rich regimen may prevent the development and the progress of colon cancer.
BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium c... more BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhαIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect α-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8–10, 19–22, and 38–43. To evaluate their functional role, three BotXIV/LqhαIT chimeras were designed by transferring the corresponding LqhαIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular α/β fold. Interestingly, substitution of residues 8–10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of α-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure–function relationships between insect α-toxins and α-like scorpion toxins are probably more complex than expected.
Toxicon : official journal of the International Society on Toxinology, 1997
One contractive and two depressant toxins active on insect were purified by high-performance liqu... more One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = ...
We purified to homogeneity from venom of Buthus occitanus tunetanus seven toxins active on the co... more We purified to homogeneity from venom of Buthus occitanus tunetanus seven toxins active on the cockroach Periplaneta germanica. These toxins belong to previously defined groups, contracturant and depressive relaxant. A dose of 50-150 ng of toxins is able to kill 50% of injected P. germanica but only the contracturant toxins show toxicity to C57B16 mice injected i.c.v, with a DLs0 of 1 #g. The binding of 125I AaHIT (an excitatory contracturant toxin) to P. americana synaptosomes is inhibited at the same level as AaHIT, by the following depressive toxins: BotlT4, BotlT5 and BotlT6 and also by a contracturant toxin BotlT2. The half-effect is about 1-4 nM. 10 -6 M of delayed contracturant toxins BotlTl, BotIT3 and BotlT7 did not inhibit the binding of t25I AaHIT to its receptor. BotlT1, BotlT2, BotlT3 and BotlT7 cross-react with AaHIT as observed in ELISA tests using anti-AaHIT antibodies. These toxins are not recognized by anti-LqqlT2 antibodies. BotlT4 and BotlT5, which differ by a unique substitution in position 6, Lys instead of Arg, show about 80% sequence homology with LqqlT2. In contrast, BotlT4, BotlT5 and BotlT6 are recognized exclusively by anti-LqqlT2 antibodies. Purified Bot insect toxins, when applied on the isolated giant axon of the cockroach P. americana, show various effects. BotlT4, BotlT5 and BotlT6 are able to block the action potential and progressively suppress the Na ÷ current. BotlT1, BotlT3 and BotlT7 prolong the inactivation phase of the action potential, resulting in a large augmentation of Na ÷ current. BotlT2 induces limited depolarization concomitantly with the development of depolarization after potential, repetitive activity and later plateau potentials terminated by bursts.
A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Per... more A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetunus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and a-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As a-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike a-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na' channels by decreasing the peak Na+current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na+-channel activation.
Uploads
Papers by Habib Karoui