Taurolidine单独或联合X射线抑制黑色素瘤生长及其机制
摘要
Taurolidine作为抗生素和抗毒素类药物一直在临床上治疗外科手术后感染及腹腔粘连。近几年来,许多实验室报道,taurolidine具有诱导多种肿瘤细胞凋亡及抑制其生长的特性。本研究选择2种鼠黑色素瘤细胞(B16-4A5和B16-F10)和2种人黑色素瘤细胞(WM-793和1205-Lu),进一步探讨其抑瘤效应及作用机制,并首次报道taurolidine联合X射线照射,对黑色素瘤细胞具有协同抑瘤作用。本实验中,taurolidine对上述4种黑色素瘤细胞均显示了强烈的细胞毒效应,体内实验进一步表明,taurolidine通过诱导小鼠B16-4A5细胞凋亡,抑制局部肿瘤生长; taurolidine提高小鼠CTL和NK细胞的细胞毒效应和抑制TNF-α的活性,可能是其抑制小鼠黑色素瘤局部生长和远处转移的另一发生机制。在taurolidine诱导B16-4A5细胞凋亡的机制性评价中,本研究发现taurolidine通过提高促凋亡蛋白Bad和Bax的表达和抑制抗凋亡蛋白Bcl-2的表达,激活caspase-9和caspase-3,通过线粒体途径诱导小鼠黑色素瘤B16-4A5细胞凋亡。在taurolidine联合放疗的实验中,体内和体外研究发现taurolidinel可提高2种鼠黑色素瘤细胞的放射敏感性,提高局部抑瘤效果;然而,对2种人的黑色素瘤细胞,联合治疗未显示出协同的治疗作用。在taurolidine提高放射敏感性机制性的评价中,发现taurolidine联合X射线可协同诱导促凋亡蛋白Bad和Bax的表达,抑制抗凋亡蛋白Bcl-2的表达,并提高caspase-9的活性,从而说明联合治疗通过对凋亡传导通路信号的调控,提高放射敏感性。细胞周期评价,显示taurolidine联合X射线照射后, G2/M期阻滞的B16-4A5和B16-F10细胞显著减少,而WM-793和1205-Lu细胞进一步发生G2/M期阻滞,说明联合治疗导致G2/M期阻滞的去除,可能是taurolidine提高小鼠黑色素瘤细胞放射敏感性的另一发生机制。本研究为taurolidine作为抗肿瘤药物应用临床提供科学依据;然而,是否可作为放射增敏剂应用临床,仍需进一步的研究。
To date, it is best known for its antibacterial effect since taurolidine was designed in the 1970s as a broad-apectrum. In the clinic setting, taurolidine has been used as an anti-bacterial agent in patients with established peritonitis and with inflammatory response syndrome. Recently, a growing body of evidence supports taurolidine as an anti-neoplastic agent. Further studies show that taurolidine inhibits tumor cell growth mainly by induction of apoptosis, but it is very hard for taurolidine to induce apoptosis in the normal bone fiber cells. The more interesting founding is that taurolidine causes a permitted toxicity in blood cells, and liver and kidney founction. However, it is not quite clear for the mechanistic studies of inhibiting tumor growth induced by taurolidine. Some studies showed that taurolidine inhibited the proliferation in human prostate tumor cells through mitochondrial cytochrome-c dependent pathway of apoptosis. Other studies reported that taurolidine was found to have direct and selected anti-neoplastic effects on glial and neuronal brain tumor cells via Fas ligand-mediated programmed cell death. So further mechanistic studies are necessary for taurolidine to evaluate its antineoplastic effects.
Radiotherapy plays a very important role in the treatment of malignancies, but its application is currently limited by the side-effects such as the radiation-induced damages in the normal tissues nearby and the radiation-resistance to certain tumors. To solve these problems, effective agents need to be found to enhance radiosensitivity and futher inhibit local tumor growth. Melanoma has become an important public health issue because of its rising incidence around the world. Due to hard induction of apoptosis, malignant melanoma is characterized by poorly responds to conventional chemotherapy and radiotherapy. We hypothesize that taurolidine can induce apoptosis in malignant melanoma cells and trigger radiosensitivity to enhance irradiation-induced cytotocity. We now report that the treatment of murine B16-4A5 and B16-F10 and human WM-793 and 1205-Lu melanoma cells with taurolidine in vitro induced cell apoptosis and decreased cell viability. In vivo systemic administration of taurolidine showed a significant attenuation of primary and metastatic tumor growth in murine melanoma B16-4A5 tumor-bearing mice, which was associated with an increased intra-tumor apoptosis and enhanced NK, CTL and a reduced TNF-αactivity. Its mechanisms of induction of apoptosis may be associated with the mediation of Bcl-2 family protein and activation of mitocontrial pathway. Meanwhile, we first report that administration of taurolidine can trigger radiosensitivity in B16-4A5 and B16-F10 cells and show a synergistic attenuation of local primary tumor growth in vivo when combined with irradiation. Mechanistic studies of taurolidine-enhanced radiosensitivity in B16-4A5 cells showed that combination treatment further increased the levels of proapototic protein Bax and Bad and reduced the levels of antiapoptotic protein Bcl-2, when compared with either agent alone, which suggestted that Bcl-2 family might be involved in the mechanisms of taurolidine-enhanced radiosensitivity. The decline of cell cycle arrest in G2/M induced by combination therapy could be another mechanism of radiosensitivity enhancement in two murine melanoma cells. However, combination therapy had not shown synergistic effect on both human melamona cell lines. These data suggest the potential of taurolidine as a novel anti-tumor agent for malignant melanoma. But further studies are still required to evaluate its synergistic effect on human melanoma cells if as an irradiation trigger in the clinic.
1 Attenuation of tumor growth in vitro and in vivo induced by taurolidine and it mechanisms
1.1 Effect of taurolidine on cell viability in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 96-well, flat-bottom, culture plates at 1×104 cells/well. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) at 37oC in humidified 5% CO2 condition for 24 and 48 h. The effect of taurolidine on cell viability was assessed by MTT assay. Incubation with taurolidine at 50μM for 48 h resulted in a nearly 50% and 60% (P < 0.05) reduction in both murine and human melanoma cells, respectively, with a dose- and time-dependent manner.
1.2 Evaluation of cell apoptosis in vitro induced by taurolidine
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) at 37oC in humidified 5% CO2 condition for 24 and 48 h. Cell apoptosis was assessed according to flow cytometry analysis. Incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 12% cell apoptosis in two murine cell lines, B16-4A5 and B16-F10 cells, respectively. After cells were expored to taurolidine at 50μmol/L for 48 h, 66% and 22% cell apoptosis were observed in both human melanoma cells, respectively, with a dose- and time-dependent manner.
1.3 Effect of taurolidine on cell cycle progression in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) for 24 and 48 h. Cell cycle progression was assessed according to flow cytometry analysis. Flow cytometry revealed a prominent Sub-G1 peak in four cells, which is a hallmark of cells that are undergoing apoptosis, and a reduction of cells in S and G2/M phase in B16-4A5 cell, the most resistant phase of cell cycle to chemical drugs. However, taurolidine did not result in redistribution of cell cycle in other 3 cell lines. Above data further suggest that taurolidine-induced cytotoxity are via induction of cell apoptosis and necrosis.
1.4 Mechanistic evaluation of cell apoptosis induced by taurolidine
1.4.1 The proteins of Bcl-2 family, such as pro-apoptotic protein (Bax or Bad) and anti-apoptotic protein (Bcl-2), play crucial roles in regulation of apoptosis. As taurolidne-induced apoptosis can be found at 24 h, we selected 3, 6, 12 and 24 h after treatment with taurolidne for further mechanism studies. Using Western blot analysis, we found that the treatment with taurolidine results in a time-dependent increase in the levels of pro-apoptotic protein Bax and Bad, whereas the anti-apoptotic protein Bcl-2 was correspondingly reduced with a time-dependent manner.
1.4.2 Pro-apoptotic members, such as Bax and Bad, lead to the penetration of mitochondrial membrane and activation of caspase-9. Active caspase-9 then activates caspase-3, which subsequently activates the rest of the caspase cascade and finally lead to apoptosis. Therefore, we next tested weather taurolidne-induced apoptosis was through mitochondrial dependent death pathway in B16-4A5 cells by the observation of expression of caspase-9 and caspase-3 and activation of caspase-9 with Western blotting and colorimetric protease assay. The results show that taurolidine results in a dose-dependent increase in the level of caspase-9 and caspase-3. The treatment of B16-4A5 cells with taurolidine (0, 25 and 50μM) for 24 h resulted in a dose-dependent increase in the activation of caspase-9 (from 0.07 to 0.3 in A value at 405 nm), which suggest that taurolidine-induced apoptosis is associated with the mediation of Bcl-2 family, the activation of caspase-9 and caspase-3.
1.4.3 Another pathway of apoptosis is via the induction of death receptor such as Fas-ligand, which activates caspase-8 or caspase-10 to motivate caspase cascade. Thus, the level of Fas-ligand protein and the activation of caspas-8 are evaluated by Western blotting and colorimetric protease assay. The results showed that the treatment with taurolidine did not result in the increase of Fas-ligand protein and the activation of caspase-8, which further suggest that taurolidine-induced apoptosis is through mitochondrial dependent death pathway.
1.5 Evaluation of primary and metastatic tumor growth attenuated by taurolidine in vivo
1.5.1 The evaluation of cytotoxity induced by taurolidine was first evaluated in C57BL/6J mice. Our findings that taurolidine appeared to induce apoptosis in vitro were tested in the mice bearing murine B16-4A5 tumor xenografts. Initial studies were designed to identify the maximally tolerated dose of 7 consecutive days i.p. bolus injection of taurolidine. The doses ranged from 5 to 20 mg/mouse. The results of these studies revealed that doses below 15 mg/mouse/day were well tolerated. Body weight loss as a result of these dose regimens was≤9% and the mortality was below 10%. However, the dose of 20 mg/mouse/day resulted in more significant toxicity and 14% mortality.
1.5.2 Therapeutic effectiveness induced by taurolidine was next evaluated. We selected the 15-mg/mouse regimen of taurolidine for the evaluation of antineoplastic activity in the mice bearing murine B16-4A5 tumor xenografts. Taurolidine therapy was initiated on the day of tumor cell inoculation or begun 7 days thereafter, respectively. Three weeks after the initial taurolidine therapy, the mice were sacrificed, and tumors were removed. The results of this study revealed that, when initiated on the day of tumor cell injection, taurolidine therapy was highly effective to inhibit tumor growth. The mean tumor size on days 21 after taurolidine therapy was 70-fold growth, whereas, the tumor size in the control was 160-fold growth as compared with the day of initiated treatment. Equally impressive, if taurolidine therapy was begun 7 days thereafter, the mean tumor size was again significantly smaller than that in the control. Contrastingly, the early taurolidine therapy was more effective to inhibit tumor growth than delayed taurolidine therapy. Taurolidine treatment also inhibited the formation of lung metastatic nodules. However, there was no significant difference to lung metastatic nodules between the early and delayed taurolidine treatment.
1.6 Possible mechanism of primary and metastatic tumor growth attenuated by taurolidine
1.6.1 To assess apoptosis inducted by taurolidine in vivo, we performed TUNEL staining on tumor sections. The brown color indicated apoptotic nuclei as visualized using the DAB substrate. Apoptotic cells were counted under a light microscope in randomly chosen fields, and the cells in taurolidine treatment group exhibited abundant brown apoptotic nuclei, which were very few in the control.
1.6.2 The effect of taurolidine on anti-tumor immune response was assessed by the measurement of splenic CTL- and NK cell-mediated cytotoxicities to B16-4A5 and YAC-1 cells, and TNF-αreleased by peritoneal macrophages. An increased granzyme B-positive B16-4A5 and YAC-1 cells were observed and taurolidine treatment also induced a decreased TNF-αrelease.
2 Synergistic anti-tumor effect of taurolidine in combination with irradiation on melanoma cells and it mechanism
2.1 Effect of taurolidine in combination with irradiation on cell viability in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 96-well, flat-bottom, culture plates at 1×104 cells/well. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in combination with X-ray irradiation at the does of 0.5, 1, 2 and 4 Gy for 24 and 48 h. The effect of combination treatment on cell viability was assessed by MTT assay. The incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 50% a reduction in two murine melanoma cells with a dose- and time-dependent manner. Furthermore, the combination of taurolidine at 50μmol/L with X-ray irradiation at 4 Gy resulted in a nearly 80% reduction in cell viability. However, combination treatment did not result in synergistic effect on cell viability in two human cells.
2.2 Effect of taurolidine in combination with irradiation on cell apoptosis in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in the combination with X-ray irradiation at the does of 0.5, 1, 2, and 4 Gy for 24 and 48 h. Cell apoptosis was assessed according to flow cytometry analysis. The incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 12% cell apoptosis in two murine cell lines, fuethermore, a 25% cell apoptosis was further induced after the combination of taurolidine at 50μmol/L with X-ray irradiation at 4 Gy. The combination treatment also did not result in synergistic effect on cell apoptosis in two human cells.
2.3 Radiosensitivity triggered by different concentration of taurolidine in vivo
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 25-cm2 cell culture flasks at 1×104 cells/flask. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in the combination with X-ray irradiation at the does of 0, 2, 4, 6, 8 and 10 Gy for 72 h. The cells were then rinsed with PBS and allowed to grow in drug-free medium for 6 days to format colonies and then stained with 0.5% crystal violet in absolute methanol. The colonies were counted visually with a cutoff value of > 50 viable cells. Survival fraction curves calculated using the single-hit multi-target (SHMT) model: S/S0 = 1– (1– e–D/D0)n, where S/S0 is the survival fraction and D is the dose (Gy). Experiments were repeated in triplicate with each treatment. The results showed that taurolidine was determined to augment the effects of radiation in the B16-4A5 and B16-F10 cells, and the SER D0 values were determined to be 1.03 to 2.0 and 1.05 to 1.78, respectively. However, taurolidine exerted no irradiation-enhancing effects in the WM-793 and 1205-Lu cells.
2.4 Possible mechanism of taurolidine-enhanced radiosensitivity
2.4.1 It has been reported that tumor cells are resistent to apoptosis induced by irradiation mainly relying on the high level of anti-apoptotic protein, such as Bcl-XL or Bcl-2. Thus, the possible mechanisms of taurolidine-enhanced radiosensitivity were investigated in B16-4A5 cells. After cells were exposed to either irradiation at 4 Gy or in combination with the different concentration of taurolidine at 0, 10, 25 and 50μmol/L for 12 h, the expressions of Bcl-2 family proteins were tested by Western blotting assay. The results showed that, as compared with either taurolidine or irradiation alone, the combination treatment caused a more evident increase in the level of proapototic protein Bax or Bad and a reduce in the level of antiapoptotic protein Bcl-2.
2.4.2 The activation of caspase-9 was assessed with colorimetric protease assay. Comparing with either taurolidine or irradiation alone, the combination treatment resulted in a more evident increase in the level of activation of caspase-9. These data suggest that taurolidine enhanced radiosensitivity maily via the mediation of Bcl-2 family, the activation of caspas-9 and the motivation of finally apoptosis pathway.
2.4.3 The redistribution of cell cycle induced by the combination therapy was evaluated in four melonamon cells with flow cytometry assay. The irradiation alone showed a dose-dependent arrest of G2/M phase in B16-4A and B16-F10 cells, whereas a dose-dependent decline of cells in G2/M phase was found after the combination of taurolidine with irradiation. The irradiation alone showed also a dose-dependent arrest of G2/M phase in WM-793 and 1205-Lu cells, and a further accumulation of cells in G2/M phase was observed after the combination treatment. These data suggest that a dose-dependent decline of cells in G2/M phase induced by the combination treatment may be another mechanism of taurolidine-enhanced radiosnesitivity.
2.5 Primary and metastatic tumor growth in vivo attenuated by taurolidine in combination with irradiation
2.5.1 The right flank of C57Bl/6 mice was subcutaneously injected with 5×105 B16-4A5 cells suspended in 100μl PBS. Immediately thereafter, the mice were randomly divided into one of the following five groups (n = 10 mice per group). The mice at group 1 received PBS as control, the mice at group 2 and 3 received injection of i.p. bolus of 5% PVP and 5 mg/mouse TRD, while the mice at group 4 and 5 received local X-ray irradiation (10 Gy) after exposure with 5% PVP and 5 mg/mouse TRD. Primary tumor size was measured in alternate days. 14 days after the initiation of treatment, all animals were sacrificed and weighted. The results of this study revealed that a significantly attenuation of tumor growth rate was observed in mice received taurolidine alone as early as day 7. Moreover, a futher attenuation of tumor rowth rate initiated from day 5 was observed in the mice received a combination of taurolidine with local irradiation, whereas the mice received irradiation alone showed no significant inhibitory effect on flank tumor growth. Atttenuated primary tumor gwoth was further confirmed by significantly reduced tumor/body ratio in the mice received tauroidine plus irradiation group when compared with either taurolidine or irradiation alone. However, a combination of taurolidine with irradiation did not lead a further reduction in lung metasttatic tumor nodules
2.5.2 TUNEL assay for in situ apoptosis detection: Tthe mice were randomly divided into four group: (1) control; (2) taurolidine (5 mg/mouse); (3) irridiation alone; (4) taurolidine plus irradiation (5 mg + 10 Gy/mouse). As a result, the apoptotic cells exhibited brown nuclear staining, whereas counterstaining control showed a pale green staining of all cells. The apoptotic cells were evaluated under double-blinded conditions and the mean number of apoptotic cells per 15 high power fields was calculated for each experimental group. The results revealed that the cells the combination of taurolidine with irradiation exhibited much abundant brown apoptotic nuclei when compared with either taurolidine or control group alone.
2.5.3 The assays for splenic NK and CTL activity and for the release of TNF-αperitoneal macrophage: The results showed that the combination treatment did not further enhance cytotoxicity of NK and CTL cells and the decrease of TNF-αinduced by taurolidine plus irradiation.
To date, it is best known for its antibacterial effect since taurolidine was designed in the 1970s as a broad-apectrum. In the clinic setting, taurolidine has been used as an anti-bacterial agent in patients with established peritonitis and with inflammatory response syndrome. Recently, a growing body of evidence supports taurolidine as an anti-neoplastic agent. Further studies show that taurolidine inhibits tumor cell growth mainly by induction of apoptosis, but it is very hard for taurolidine to induce apoptosis in the normal bone fiber cells. The more interesting founding is that taurolidine causes a permitted toxicity in blood cells, and liver and kidney founction. However, it is not quite clear for the mechanistic studies of inhibiting tumor growth induced by taurolidine. Some studies showed that taurolidine inhibited the proliferation in human prostate tumor cells through mitochondrial cytochrome-c dependent pathway of apoptosis. Other studies reported that taurolidine was found to have direct and selected anti-neoplastic effects on glial and neuronal brain tumor cells via Fas ligand-mediated programmed cell death. So further mechanistic studies are necessary for taurolidine to evaluate its antineoplastic effects.
Radiotherapy plays a very important role in the treatment of malignancies, but its application is currently limited by the side-effects such as the radiation-induced damages in the normal tissues nearby and the radiation-resistance to certain tumors. To solve these problems, effective agents need to be found to enhance radiosensitivity and futher inhibit local tumor growth. Melanoma has become an important public health issue because of its rising incidence around the world. Due to hard induction of apoptosis, malignant melanoma is characterized by poorly responds to conventional chemotherapy and radiotherapy. We hypothesize that taurolidine can induce apoptosis in malignant melanoma cells and trigger radiosensitivity to enhance irradiation-induced cytotocity. We now report that the treatment of murine B16-4A5 and B16-F10 and human WM-793 and 1205-Lu melanoma cells with taurolidine in vitro induced cell apoptosis and decreased cell viability. In vivo systemic administration of taurolidine showed a significant attenuation of primary and metastatic tumor growth in murine melanoma B16-4A5 tumor-bearing mice, which was associated with an increased intra-tumor apoptosis and enhanced NK, CTL and a reduced TNF-αactivity. Its mechanisms of induction of apoptosis may be associated with the mediation of Bcl-2 family protein and activation of mitocontrial pathway. Meanwhile, we first report that administration of taurolidine can trigger radiosensitivity in B16-4A5 and B16-F10 cells and show a synergistic attenuation of local primary tumor growth in vivo when combined with irradiation. Mechanistic studies of taurolidine-enhanced radiosensitivity in B16-4A5 cells showed that combination treatment further increased the levels of proapototic protein Bax and Bad and reduced the levels of antiapoptotic protein Bcl-2, when compared with either agent alone, which suggestted that Bcl-2 family might be involved in the mechanisms of taurolidine-enhanced radiosensitivity. The decline of cell cycle arrest in G2/M induced by combination therapy could be another mechanism of radiosensitivity enhancement in two murine melanoma cells. However, combination therapy had not shown synergistic effect on both human melamona cell lines. These data suggest the potential of taurolidine as a novel anti-tumor agent for malignant melanoma. But further studies are still required to evaluate its synergistic effect on human melanoma cells if as an irradiation trigger in the clinic.
1 Attenuation of tumor growth in vitro and in vivo induced by taurolidine and it mechanisms
1.1 Effect of taurolidine on cell viability in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 96-well, flat-bottom, culture plates at 1×104 cells/well. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) at 37oC in humidified 5% CO2 condition for 24 and 48 h. The effect of taurolidine on cell viability was assessed by MTT assay. Incubation with taurolidine at 50μM for 48 h resulted in a nearly 50% and 60% (P < 0.05) reduction in both murine and human melanoma cells, respectively, with a dose- and time-dependent manner.
1.2 Evaluation of cell apoptosis in vitro induced by taurolidine
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) at 37oC in humidified 5% CO2 condition for 24 and 48 h. Cell apoptosis was assessed according to flow cytometry analysis. Incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 12% cell apoptosis in two murine cell lines, B16-4A5 and B16-F10 cells, respectively. After cells were expored to taurolidine at 50μmol/L for 48 h, 66% and 22% cell apoptosis were observed in both human melanoma cells, respectively, with a dose- and time-dependent manner.
1.3 Effect of taurolidine on cell cycle progression in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine (final concentration: 0, 25, 50, 100 and 200μmol/L) for 24 and 48 h. Cell cycle progression was assessed according to flow cytometry analysis. Flow cytometry revealed a prominent Sub-G1 peak in four cells, which is a hallmark of cells that are undergoing apoptosis, and a reduction of cells in S and G2/M phase in B16-4A5 cell, the most resistant phase of cell cycle to chemical drugs. However, taurolidine did not result in redistribution of cell cycle in other 3 cell lines. Above data further suggest that taurolidine-induced cytotoxity are via induction of cell apoptosis and necrosis.
1.4 Mechanistic evaluation of cell apoptosis induced by taurolidine
1.4.1 The proteins of Bcl-2 family, such as pro-apoptotic protein (Bax or Bad) and anti-apoptotic protein (Bcl-2), play crucial roles in regulation of apoptosis. As taurolidne-induced apoptosis can be found at 24 h, we selected 3, 6, 12 and 24 h after treatment with taurolidne for further mechanism studies. Using Western blot analysis, we found that the treatment with taurolidine results in a time-dependent increase in the levels of pro-apoptotic protein Bax and Bad, whereas the anti-apoptotic protein Bcl-2 was correspondingly reduced with a time-dependent manner.
1.4.2 Pro-apoptotic members, such as Bax and Bad, lead to the penetration of mitochondrial membrane and activation of caspase-9. Active caspase-9 then activates caspase-3, which subsequently activates the rest of the caspase cascade and finally lead to apoptosis. Therefore, we next tested weather taurolidne-induced apoptosis was through mitochondrial dependent death pathway in B16-4A5 cells by the observation of expression of caspase-9 and caspase-3 and activation of caspase-9 with Western blotting and colorimetric protease assay. The results show that taurolidine results in a dose-dependent increase in the level of caspase-9 and caspase-3. The treatment of B16-4A5 cells with taurolidine (0, 25 and 50μM) for 24 h resulted in a dose-dependent increase in the activation of caspase-9 (from 0.07 to 0.3 in A value at 405 nm), which suggest that taurolidine-induced apoptosis is associated with the mediation of Bcl-2 family, the activation of caspase-9 and caspase-3.
1.4.3 Another pathway of apoptosis is via the induction of death receptor such as Fas-ligand, which activates caspase-8 or caspase-10 to motivate caspase cascade. Thus, the level of Fas-ligand protein and the activation of caspas-8 are evaluated by Western blotting and colorimetric protease assay. The results showed that the treatment with taurolidine did not result in the increase of Fas-ligand protein and the activation of caspase-8, which further suggest that taurolidine-induced apoptosis is through mitochondrial dependent death pathway.
1.5 Evaluation of primary and metastatic tumor growth attenuated by taurolidine in vivo
1.5.1 The evaluation of cytotoxity induced by taurolidine was first evaluated in C57BL/6J mice. Our findings that taurolidine appeared to induce apoptosis in vitro were tested in the mice bearing murine B16-4A5 tumor xenografts. Initial studies were designed to identify the maximally tolerated dose of 7 consecutive days i.p. bolus injection of taurolidine. The doses ranged from 5 to 20 mg/mouse. The results of these studies revealed that doses below 15 mg/mouse/day were well tolerated. Body weight loss as a result of these dose regimens was≤9% and the mortality was below 10%. However, the dose of 20 mg/mouse/day resulted in more significant toxicity and 14% mortality.
1.5.2 Therapeutic effectiveness induced by taurolidine was next evaluated. We selected the 15-mg/mouse regimen of taurolidine for the evaluation of antineoplastic activity in the mice bearing murine B16-4A5 tumor xenografts. Taurolidine therapy was initiated on the day of tumor cell inoculation or begun 7 days thereafter, respectively. Three weeks after the initial taurolidine therapy, the mice were sacrificed, and tumors were removed. The results of this study revealed that, when initiated on the day of tumor cell injection, taurolidine therapy was highly effective to inhibit tumor growth. The mean tumor size on days 21 after taurolidine therapy was 70-fold growth, whereas, the tumor size in the control was 160-fold growth as compared with the day of initiated treatment. Equally impressive, if taurolidine therapy was begun 7 days thereafter, the mean tumor size was again significantly smaller than that in the control. Contrastingly, the early taurolidine therapy was more effective to inhibit tumor growth than delayed taurolidine therapy. Taurolidine treatment also inhibited the formation of lung metastatic nodules. However, there was no significant difference to lung metastatic nodules between the early and delayed taurolidine treatment.
1.6 Possible mechanism of primary and metastatic tumor growth attenuated by taurolidine
1.6.1 To assess apoptosis inducted by taurolidine in vivo, we performed TUNEL staining on tumor sections. The brown color indicated apoptotic nuclei as visualized using the DAB substrate. Apoptotic cells were counted under a light microscope in randomly chosen fields, and the cells in taurolidine treatment group exhibited abundant brown apoptotic nuclei, which were very few in the control.
1.6.2 The effect of taurolidine on anti-tumor immune response was assessed by the measurement of splenic CTL- and NK cell-mediated cytotoxicities to B16-4A5 and YAC-1 cells, and TNF-αreleased by peritoneal macrophages. An increased granzyme B-positive B16-4A5 and YAC-1 cells were observed and taurolidine treatment also induced a decreased TNF-αrelease.
2 Synergistic anti-tumor effect of taurolidine in combination with irradiation on melanoma cells and it mechanism
2.1 Effect of taurolidine in combination with irradiation on cell viability in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 96-well, flat-bottom, culture plates at 1×104 cells/well. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in combination with X-ray irradiation at the does of 0.5, 1, 2 and 4 Gy for 24 and 48 h. The effect of combination treatment on cell viability was assessed by MTT assay. The incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 50% a reduction in two murine melanoma cells with a dose- and time-dependent manner. Furthermore, the combination of taurolidine at 50μmol/L with X-ray irradiation at 4 Gy resulted in a nearly 80% reduction in cell viability. However, combination treatment did not result in synergistic effect on cell viability in two human cells.
2.2 Effect of taurolidine in combination with irradiation on cell apoptosis in vitro
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 6-well,flat-bottom,culture plates. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in the combination with X-ray irradiation at the does of 0.5, 1, 2, and 4 Gy for 24 and 48 h. Cell apoptosis was assessed according to flow cytometry analysis. The incubation with taurolidine at 50μmol/L for 48 h resulted in a nearly 12% cell apoptosis in two murine cell lines, fuethermore, a 25% cell apoptosis was further induced after the combination of taurolidine at 50μmol/L with X-ray irradiation at 4 Gy. The combination treatment also did not result in synergistic effect on cell apoptosis in two human cells.
2.3 Radiosensitivity triggered by different concentration of taurolidine in vivo
Both of murine and human malonoma cells (B16-4A5,B16-F10,WM-793 and 1205-Lu cells) were plated onto 25-cm2 cell culture flasks at 1×104 cells/flask. The cells were treated with taurolidine at the concentration of 0, 10, 25 and 50μmol/L in the combination with X-ray irradiation at the does of 0, 2, 4, 6, 8 and 10 Gy for 72 h. The cells were then rinsed with PBS and allowed to grow in drug-free medium for 6 days to format colonies and then stained with 0.5% crystal violet in absolute methanol. The colonies were counted visually with a cutoff value of > 50 viable cells. Survival fraction curves calculated using the single-hit multi-target (SHMT) model: S/S0 = 1– (1– e–D/D0)n, where S/S0 is the survival fraction and D is the dose (Gy). Experiments were repeated in triplicate with each treatment. The results showed that taurolidine was determined to augment the effects of radiation in the B16-4A5 and B16-F10 cells, and the SER D0 values were determined to be 1.03 to 2.0 and 1.05 to 1.78, respectively. However, taurolidine exerted no irradiation-enhancing effects in the WM-793 and 1205-Lu cells.
2.4 Possible mechanism of taurolidine-enhanced radiosensitivity
2.4.1 It has been reported that tumor cells are resistent to apoptosis induced by irradiation mainly relying on the high level of anti-apoptotic protein, such as Bcl-XL or Bcl-2. Thus, the possible mechanisms of taurolidine-enhanced radiosensitivity were investigated in B16-4A5 cells. After cells were exposed to either irradiation at 4 Gy or in combination with the different concentration of taurolidine at 0, 10, 25 and 50μmol/L for 12 h, the expressions of Bcl-2 family proteins were tested by Western blotting assay. The results showed that, as compared with either taurolidine or irradiation alone, the combination treatment caused a more evident increase in the level of proapototic protein Bax or Bad and a reduce in the level of antiapoptotic protein Bcl-2.
2.4.2 The activation of caspase-9 was assessed with colorimetric protease assay. Comparing with either taurolidine or irradiation alone, the combination treatment resulted in a more evident increase in the level of activation of caspase-9. These data suggest that taurolidine enhanced radiosensitivity maily via the mediation of Bcl-2 family, the activation of caspas-9 and the motivation of finally apoptosis pathway.
2.4.3 The redistribution of cell cycle induced by the combination therapy was evaluated in four melonamon cells with flow cytometry assay. The irradiation alone showed a dose-dependent arrest of G2/M phase in B16-4A and B16-F10 cells, whereas a dose-dependent decline of cells in G2/M phase was found after the combination of taurolidine with irradiation. The irradiation alone showed also a dose-dependent arrest of G2/M phase in WM-793 and 1205-Lu cells, and a further accumulation of cells in G2/M phase was observed after the combination treatment. These data suggest that a dose-dependent decline of cells in G2/M phase induced by the combination treatment may be another mechanism of taurolidine-enhanced radiosnesitivity.
2.5 Primary and metastatic tumor growth in vivo attenuated by taurolidine in combination with irradiation
2.5.1 The right flank of C57Bl/6 mice was subcutaneously injected with 5×105 B16-4A5 cells suspended in 100μl PBS. Immediately thereafter, the mice were randomly divided into one of the following five groups (n = 10 mice per group). The mice at group 1 received PBS as control, the mice at group 2 and 3 received injection of i.p. bolus of 5% PVP and 5 mg/mouse TRD, while the mice at group 4 and 5 received local X-ray irradiation (10 Gy) after exposure with 5% PVP and 5 mg/mouse TRD. Primary tumor size was measured in alternate days. 14 days after the initiation of treatment, all animals were sacrificed and weighted. The results of this study revealed that a significantly attenuation of tumor growth rate was observed in mice received taurolidine alone as early as day 7. Moreover, a futher attenuation of tumor rowth rate initiated from day 5 was observed in the mice received a combination of taurolidine with local irradiation, whereas the mice received irradiation alone showed no significant inhibitory effect on flank tumor growth. Atttenuated primary tumor gwoth was further confirmed by significantly reduced tumor/body ratio in the mice received tauroidine plus irradiation group when compared with either taurolidine or irradiation alone. However, a combination of taurolidine with irradiation did not lead a further reduction in lung metasttatic tumor nodules
2.5.2 TUNEL assay for in situ apoptosis detection: Tthe mice were randomly divided into four group: (1) control; (2) taurolidine (5 mg/mouse); (3) irridiation alone; (4) taurolidine plus irradiation (5 mg + 10 Gy/mouse). As a result, the apoptotic cells exhibited brown nuclear staining, whereas counterstaining control showed a pale green staining of all cells. The apoptotic cells were evaluated under double-blinded conditions and the mean number of apoptotic cells per 15 high power fields was calculated for each experimental group. The results revealed that the cells the combination of taurolidine with irradiation exhibited much abundant brown apoptotic nuclei when compared with either taurolidine or control group alone.
2.5.3 The assays for splenic NK and CTL activity and for the release of TNF-αperitoneal macrophage: The results showed that the combination treatment did not further enhance cytotoxicity of NK and CTL cells and the decrease of TNF-αinduced by taurolidine plus irradiation.
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