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Combining a matrix metalloproteinase inhibitor, a farnesyltransferase inhibitor, and a taxane improves survival in an anaplastic thyroid cancer model
Cancer Letters 238 (2006) 197–201 www.elsevier.com/locate/canlet
Combining a matrix metalloproteinase inhibitor, a farnesyltransferase inhibitor, and a taxane improves survival in an anaplastic thyroid cancer model Miaorong Shea,#, Sai-Ching Jim Yeunga,b,* a
Department of General Internal Medicine, Ambulatory Treatment and Emergency Care, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA b Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA Received 30 October 2004; accepted 5 July 2005
Abstract We previously showed that the in vivo anticancer effects of a combination of manumycin (a farnesyltransferase inhibitor) and paclitaxel (a microtubule inhibitor) against anaplastic thyroid carcinoma (ATC) were partially due to inhibition of angiogenesis. In this study, we investigated the effect of adding minocycline (a matrix metalloproteinase inhibitor) to manumycin and paclitaxel against human ATC cells xenografted in nude mice. The triple-drug combination resulted in the lowest average tumor growth rate, and it conferred significantly better survival than manumycin alone, paclitaxel alone, or manumycin plus paclitaxel. In conclusion, this novel combination deserves further investigation in the treatment of ATC. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Manumycin; Paclitaxel; Minocycline; Human anaplastic thyroid carcinoma; Nude mouse xenograft model; Angiogenesis inhibition
1. Introduction Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors, and patients with ATC have a very poor prognosis, with a mean survival of * Corresponding author. Address: The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 437, Houston, TX 77030, USA. Tel.: C1 713 745 4516. E-mail address: [email protected] (S.-C. Jim Yeung). # Dr. M. She is a visiting scholar from the Department of Hematology, Guangdong Provincial People’s Hospital, Guangzhou, People’s Republic of China.
3–7 months that is not improved very much by current therapy [1]. Our group has demonstrated the antineoplastic effect of manumycin A (manumycin), a farnesyltransferase inhibitor, against ATC and the enhancement of apoptosis by the addition of paclitaxel (a taxane inhibitor of microtubule function) [2]. Our preliminary results also indicated that angiogenesis inhibition mediated, at least partially, the in vivo antineoplastic effect of manumycin plus paclitaxel [3]. Two other recent reports have also provided evidence that farnesyltransferase inhibitors inhibit tumor angiogenesis [4,5].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.07.012
198
M. She, S.-C. Jim Yeung / Cancer Letters 238 (2006) 197–201
Solid tumors depend on angiogenesis for their growth. Matrix metalloproteinases (MMPs) are an important group of enzymes mediating the endothelial cell invasion and migration required in the formation of new capillaries. Minocycline is a semisynthetic analogue of tetracycline activity of MMPs through chelation of the zinc ion at the active site of the enzyme. We hypothesized that a triple-drug combination consisting of manumycin and paclitaxel plus the MMP inhibitor minocycline would improve antineoplastic activity over that of the single agents alone or the combination of the two drugs. Therefore, we compared the in vivo antineoplastic effect of this triple-drug combination with that of manumycin alone, paclitaxel alone, and manumycin plus paclitaxel in a 2!2!2 design in nude mice bearing ATC xenografts.
2. Materials and methods 2.1. Drugs Manumycin A and paclitaxel were purchased from Sigma (St. Louis, MO). They were dissolved in dimethylsulfoxide (DMSO) (Sigma) at appropriate concentrations prior to dilution in tissue-culture medium such that the final concentration of DMSO would not exceed 0.1% (vol/vol). Minocycline slowrelease tablets (10 mg/tablet, 60-day release period) for subcutaneous implantation and placebo tablets were purchased from Innovative Research of America (Sarasota, FL). 2.2. Cell culture A human ATC cell line, ARO, was used [6]. The cells were cultured as previously described [2].
cared for according to the NIH Guide for the Care and Use of Laboratory Animals. Sixty-four mice were randomly assigned to experimental or control groups (eight groups with eight mice per group; Table 1). Drug treatments began 6 days after xenograft implantation when all the engrafted tumors reached at least 15 mm3. Animals without wellengrafted tumors were excluded from the study. Manumycin and paclitaxel were administered in 7-day cycles for six cycles. Manumycin was injected intraperitoneally at 2.5 mg/kg/day on days 1–5 and paclitaxel was injected intraperitoneally at 50 mg/kg/ day on day 1 of each cycle. Control injections contained the identical DMSO concentration in tissue-culture medium but without the drugs. Minocycline was delivered by subcutaneous implantation of a slow-release tablet (10 mg released over 60 days) on the day of the first manumycin or paclitaxel injection. Mice that did not receive minocycline subcutaneous implants received placebo tablets. The minocycline and placebo tablets were implanted using a trochar (Innovative Research of America) at about 1 cm from the puncture sites on the back of the nude mice. The body weight, feeding behavior, and motor activity of each animal were monitored every Monday, Wednesday and Friday as indicators of general health. The tumor burden of each animal was monitored every day by the investigators as well as the veterinary medicine service of the University of Texas M. D. Anderson Cancer Center. The veterinary medicine service personnel were blinded to the treatment received by each animal. Animals with any of the following were euthanized: greater than 10% weight loss, motor retardation, inability to obtain food or Table 1 Animal treatment groups Group
Manumycin
Paclitaxel
Minocycline
Number of animals
1 (Control) 2 3 4 5 6 7 8
K C K K C C K C
K K C K C K C C
K K K C K C C C
8 8 8 8 8 8 8 8
2.3. Nude mouse xenograft model One million ARO cells suspended in RPMI 1640 medium were injected subcutaneously into a flank of each 5-week-old nude mouse (nu/nu BALB-c). Tumor volumes were calculated by the formula a2!b!0.4, where a is the smallest diameter and b is the diameter perpendicular to a [7]. The mice were housed in barrier facilities on a 12 h light/dark cycle with food and water available ad libitum. The animals were
Note: The control mice are the nude mice with ATC xenografts that were treated with placebo.
M. She, S.-C. Jim Yeung / Cancer Letters 238 (2006) 197–201
water, ruffled hair, and largest diameter of the tumor exceeding 15 mm. When the animals were euthanized, tumor xenografts were dissected and weighed. 2.4. Statistical analysis The significance of differences among multiple groups was assessed using three-way analysis of variance (Sigmastat version 3.1, Systat Software Inc.). The null hypothesis was accepted if P!0.05. The survival functions of the eight groups of animals were analyzed using the Kaplan–Meier method.
3. Results 3.1. Tumor growth rate (index) Because xenografts from different animals were dissected and obtained at different times after implantation of cancer cells and because tumor weight is a function of time after implantation, we used the tumor growth rate (or index), defined as the xenograft weight (in grams) divided by the time after tumor cell implantation (in days), to assess the effect of treatments on tumor growth. The tumor growth rates for the treatment groups are summarized in Fig. 1. The error bars represented the 95% confidence intervals. The mean tumor growth rate of the three-drug group was the lowest among all eight groups. At the 95% confidence level, the tumor growth rate of the threedrug group was significantly lower than that in the control group, the manumycin group and the manumycin plus minocycline group (one-way ANOVA, post-hoc multiple comparisons vs the three-drug group, Holm-Sidak method, P!0.05). Using three-way ANOVA (general linear model, all pairwise multiple comparison, Tukey Test) to analyze the impact on the tumor growth rate of the three factors (minocycline, manumycin and paclitaxel), we found that there was a statistically significant interaction between minocycline, manumycin and paclitaxel (PZ 0.046). The difference in the mean tumor growth rates between animals that received paclitaxel and those that did not was significant (PZ0.002). The difference in the mean tumor growth rates between those received manumycin and those that did not did not reach statistical significance (PZ0.127), and neither did the
199
difference in the mean tumor growth rates between those received minocycline and those that did not (PZ0.315). 3.2. Survival Most of the animals in this study were euthanized because of excessive tumor burden. There were significant differences in the survival of animals in the different groups (Fig. 2, Table 2). The triple-drug combination conferred the best survival. The log-rank test showed that the three-drug group had significantly (P!0.05) better survival than all the other groups except the manumycin-plus-paclitaxel group (PZ 0.0655) and the paclitaxel-plus-minocycline group (PZ0.0661). If we accept a P value of !0.10 as significant, then the three-drug group had significantly better survival than all other groups. 3.3. Toxicity The body weight of nude mice remained stable, and there were no significant differences in bodyweight changes between the groups (data not shown). The motor activity and the feeding behavior were normal. One mouse in the minocycline group died after 7 days of treatment. Another mouse in the manumycin-plus-paclitaxel group died on day 27 of drug administration.
Fig. 1. Effect of the combination of minocycline, manumycin, and paclitaxel on ARO xenograft growth. The mean tumor growth index was plotted for each treatment group. The error bars represented the 95% confidence intervals.
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Fig. 2. Effect of the combination of minocycline, manumycin, and paclitaxel on survival. Survival curves of treatment groups vs the control group. In each panel, the survival curves for the control group and a treatment group (as labeled) are shown. The horizontal axis represents the number of days since treatments began. The vertical axis represents the percentage of animals surviving.
Table 2 Survival outcomes with the various treatments Group
Median survival time (d)
95% Confidence internal
P value (vs control)
P value (vs manumycinC paclitaxelCminocycline group)
Control Manumycin Paclitaxel Minocycline ManumycinCpaclitaxel ManumycinCminocycline PaclitaxelCminocycline ManumycinCpaclitaxelCminocycline
23 28 28 23 28 22 33 46
17.29 25.31 22.34 19.27 22.34 20.24 24.42 44.48
1 0.4578 0.2888 0.7127 0.0521 0.9576 0.1204 0.001
0.001 0.0019 0.0035 0.004 0.0655 0.0127 0.0661 1
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201
4. Discussion
Acknowledgements
In this paper, we presented in vivo data from nude mice bearing ARO xenografts demonstrating that adding minocycline to the combination of manumycin and paclitaxel resulted in improved survival. At the 90% confidence level, the three-drug combination resulted in the better animal survival than any other possible combinations of the three drugs. Because each treatment group consisted of only eight animals, the power of the study was limited to the detection of large differences in any of the two-group comparisons. It has recently been suggested in the statistics literature that when only limited samples are available and a program of investigations is being undertaken, the traditional 0.05 level of significance may be relaxed and 0.10–0.20 (or higher) may be appropriate values for early-stage research [8,9]. P values between 0.05 and 0.20 were considered of interest for future investigations. Therefore, our hypothesis was supported by this experiment, and treatment with the triple-drug combination deserved further investigation in future. The tumor growth rate data were in agreement with the animal survival data. The triple combination group was the treatment group with the lowest mean tumor growth rate. Three-way ANOVA (generalized linear model analysis) demonstrated that there was a statistically significant interaction among the three drugs in their effect on the tumor growth rate. Minocycline is a safe and inexpensive drug. Its use as an antiangiogenic agent in combination with other chemotherapy for cancer has been investigated in preclinical animal models [10–12]. The combination of minocycline with a taxane and a farnesyltransferase inhibitor is novel. Although minocycline alone did not show any impressive antineoplastic effect, further exploration of its use in combination with other antineoplastic agents is warranted. In conclusion, the triple-drug combination of manumycin, paclitaxel, and minocycline was an effective combination against ATC, and resulted in improved survival in the nude mouse xenograft model. This study demonstrated the potential of a novel combination of a farnesyltransferase inhibitor, a taxane and an MMP inhibitor, which should be explored in future clinical trials.
This research was supported by a grant to SJY from Abbott Laboratories (Thyroid Research Advisory Council). The animal facility at The University of Texas M. D. Anderson Cancer Center is partially supported by the Cancer Center Support Grant (CA16672).
References [1] K.B. Ain, Anaplastic thyroid carcinoma: a therapeutic challenge, 16 (1999) 64–69. [2] S. Yeung, G. Xu, J. Pan, M. Christgen, A. Bamiagis, Manumycin enhances the cytotoxic effect of paclitaxel on anaplastic thyroid carcinoma cells, 60 (2000) 650–656. [3] G. Xu, J. Pan, C. Martin, C. Yeung, Angiogenesis inhibition in the in vivo antineoplastic effect of manumycin and paclitaxel against anaplastic thyroid carcinoma, 86 (2001) 1769–1777. [4] W.Z. Gu, S.K. Tahir, Y.C. Wang, H.C. Zhang, S.P. Cherian, S. O’Connor, J.A. Leal, S.H. Rosenberg, C. Ng, Effect of novel CAAX peptidomimetic farnesyltransferase inhibitor on angiogenesis in vitro and in vivo, 35 (1999) 1394–1401. [5] M.M. Feldkamp, N. Lau, A. Guha, Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects, 18 (1999) 7514–7526. [6] J.A. Fagin, K. Matsuo, A. Karmakar, D.L. Chen, S.H. Tang, P. Koeffler, High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas, 91 (1993) 179–184. [7] A. Yoshida, M. Fukazawa, H. Ushio, Y. Aiyoshi, S. Soeda, K. Ito, Study of cell kinetics in anaplastic thyroid carcinoma transplanted to nude mice, 41 (1989) 1–4. [8] R. Sposto, D.O. Stram, A strategic view of randomized trial design in low-incidence paediatric cancer, 18 (1999) 1183–1197. [9] N. Strauss, R. Simon, Investigating a sequence of randomized phase II trials to discover promising treatments, 14 (1995) 1479–1489. [10] E.A. Sotomayor, B.A. Teicher, G.N. Schwartz, S.A. Holden, K. Menon, T.S. Herman, E. Frei, 3rd, Minocycline in combination with chemotherapy or radiation therapy in vitro and in vivo, 30 (1992) 377–384. [11] B.A. Teicher, S.A. Holden, N.P. Dupuis, Y. Kakeji, M. Ikebe, Y. Emi, D. Goff, Potentiation of cytotoxic therapies by TNP470 and minocycline in mice bearing EMT-6 mammary carcinoma, 36 (1995) 227–236. [12] Y. Kakeji, A. Teicher, Preclinical studies of the combination of angiogenic inhibitors with cytotoxic agents, 15 (1997) 39–48.
Combining a matrix metalloproteinase inhibitor, a farnesyltransferase inhibitor, and a taxane improves survival in an anaplastic thyroid cancer model Miaorong Shea,#, Sai-Ching Jim Yeunga,b,* a
Department of General Internal Medicine, Ambulatory Treatment and Emergency Care, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA b Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA Received 30 October 2004; accepted 5 July 2005
Abstract We previously showed that the in vivo anticancer effects of a combination of manumycin (a farnesyltransferase inhibitor) and paclitaxel (a microtubule inhibitor) against anaplastic thyroid carcinoma (ATC) were partially due to inhibition of angiogenesis. In this study, we investigated the effect of adding minocycline (a matrix metalloproteinase inhibitor) to manumycin and paclitaxel against human ATC cells xenografted in nude mice. The triple-drug combination resulted in the lowest average tumor growth rate, and it conferred significantly better survival than manumycin alone, paclitaxel alone, or manumycin plus paclitaxel. In conclusion, this novel combination deserves further investigation in the treatment of ATC. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Manumycin; Paclitaxel; Minocycline; Human anaplastic thyroid carcinoma; Nude mouse xenograft model; Angiogenesis inhibition
1. Introduction Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors, and patients with ATC have a very poor prognosis, with a mean survival of * Corresponding author. Address: The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 437, Houston, TX 77030, USA. Tel.: C1 713 745 4516. E-mail address: [email protected] (S.-C. Jim Yeung). # Dr. M. She is a visiting scholar from the Department of Hematology, Guangdong Provincial People’s Hospital, Guangzhou, People’s Republic of China.
3–7 months that is not improved very much by current therapy [1]. Our group has demonstrated the antineoplastic effect of manumycin A (manumycin), a farnesyltransferase inhibitor, against ATC and the enhancement of apoptosis by the addition of paclitaxel (a taxane inhibitor of microtubule function) [2]. Our preliminary results also indicated that angiogenesis inhibition mediated, at least partially, the in vivo antineoplastic effect of manumycin plus paclitaxel [3]. Two other recent reports have also provided evidence that farnesyltransferase inhibitors inhibit tumor angiogenesis [4,5].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.07.012
198
M. She, S.-C. Jim Yeung / Cancer Letters 238 (2006) 197–201
Solid tumors depend on angiogenesis for their growth. Matrix metalloproteinases (MMPs) are an important group of enzymes mediating the endothelial cell invasion and migration required in the formation of new capillaries. Minocycline is a semisynthetic analogue of tetracycline activity of MMPs through chelation of the zinc ion at the active site of the enzyme. We hypothesized that a triple-drug combination consisting of manumycin and paclitaxel plus the MMP inhibitor minocycline would improve antineoplastic activity over that of the single agents alone or the combination of the two drugs. Therefore, we compared the in vivo antineoplastic effect of this triple-drug combination with that of manumycin alone, paclitaxel alone, and manumycin plus paclitaxel in a 2!2!2 design in nude mice bearing ATC xenografts.
2. Materials and methods 2.1. Drugs Manumycin A and paclitaxel were purchased from Sigma (St. Louis, MO). They were dissolved in dimethylsulfoxide (DMSO) (Sigma) at appropriate concentrations prior to dilution in tissue-culture medium such that the final concentration of DMSO would not exceed 0.1% (vol/vol). Minocycline slowrelease tablets (10 mg/tablet, 60-day release period) for subcutaneous implantation and placebo tablets were purchased from Innovative Research of America (Sarasota, FL). 2.2. Cell culture A human ATC cell line, ARO, was used [6]. The cells were cultured as previously described [2].
cared for according to the NIH Guide for the Care and Use of Laboratory Animals. Sixty-four mice were randomly assigned to experimental or control groups (eight groups with eight mice per group; Table 1). Drug treatments began 6 days after xenograft implantation when all the engrafted tumors reached at least 15 mm3. Animals without wellengrafted tumors were excluded from the study. Manumycin and paclitaxel were administered in 7-day cycles for six cycles. Manumycin was injected intraperitoneally at 2.5 mg/kg/day on days 1–5 and paclitaxel was injected intraperitoneally at 50 mg/kg/ day on day 1 of each cycle. Control injections contained the identical DMSO concentration in tissue-culture medium but without the drugs. Minocycline was delivered by subcutaneous implantation of a slow-release tablet (10 mg released over 60 days) on the day of the first manumycin or paclitaxel injection. Mice that did not receive minocycline subcutaneous implants received placebo tablets. The minocycline and placebo tablets were implanted using a trochar (Innovative Research of America) at about 1 cm from the puncture sites on the back of the nude mice. The body weight, feeding behavior, and motor activity of each animal were monitored every Monday, Wednesday and Friday as indicators of general health. The tumor burden of each animal was monitored every day by the investigators as well as the veterinary medicine service of the University of Texas M. D. Anderson Cancer Center. The veterinary medicine service personnel were blinded to the treatment received by each animal. Animals with any of the following were euthanized: greater than 10% weight loss, motor retardation, inability to obtain food or Table 1 Animal treatment groups Group
Manumycin
Paclitaxel
Minocycline
Number of animals
1 (Control) 2 3 4 5 6 7 8
K C K K C C K C
K K C K C K C C
K K K C K C C C
8 8 8 8 8 8 8 8
2.3. Nude mouse xenograft model One million ARO cells suspended in RPMI 1640 medium were injected subcutaneously into a flank of each 5-week-old nude mouse (nu/nu BALB-c). Tumor volumes were calculated by the formula a2!b!0.4, where a is the smallest diameter and b is the diameter perpendicular to a [7]. The mice were housed in barrier facilities on a 12 h light/dark cycle with food and water available ad libitum. The animals were
Note: The control mice are the nude mice with ATC xenografts that were treated with placebo.
M. She, S.-C. Jim Yeung / Cancer Letters 238 (2006) 197–201
water, ruffled hair, and largest diameter of the tumor exceeding 15 mm. When the animals were euthanized, tumor xenografts were dissected and weighed. 2.4. Statistical analysis The significance of differences among multiple groups was assessed using three-way analysis of variance (Sigmastat version 3.1, Systat Software Inc.). The null hypothesis was accepted if P!0.05. The survival functions of the eight groups of animals were analyzed using the Kaplan–Meier method.
3. Results 3.1. Tumor growth rate (index) Because xenografts from different animals were dissected and obtained at different times after implantation of cancer cells and because tumor weight is a function of time after implantation, we used the tumor growth rate (or index), defined as the xenograft weight (in grams) divided by the time after tumor cell implantation (in days), to assess the effect of treatments on tumor growth. The tumor growth rates for the treatment groups are summarized in Fig. 1. The error bars represented the 95% confidence intervals. The mean tumor growth rate of the three-drug group was the lowest among all eight groups. At the 95% confidence level, the tumor growth rate of the threedrug group was significantly lower than that in the control group, the manumycin group and the manumycin plus minocycline group (one-way ANOVA, post-hoc multiple comparisons vs the three-drug group, Holm-Sidak method, P!0.05). Using three-way ANOVA (general linear model, all pairwise multiple comparison, Tukey Test) to analyze the impact on the tumor growth rate of the three factors (minocycline, manumycin and paclitaxel), we found that there was a statistically significant interaction between minocycline, manumycin and paclitaxel (PZ 0.046). The difference in the mean tumor growth rates between animals that received paclitaxel and those that did not was significant (PZ0.002). The difference in the mean tumor growth rates between those received manumycin and those that did not did not reach statistical significance (PZ0.127), and neither did the
199
difference in the mean tumor growth rates between those received minocycline and those that did not (PZ0.315). 3.2. Survival Most of the animals in this study were euthanized because of excessive tumor burden. There were significant differences in the survival of animals in the different groups (Fig. 2, Table 2). The triple-drug combination conferred the best survival. The log-rank test showed that the three-drug group had significantly (P!0.05) better survival than all the other groups except the manumycin-plus-paclitaxel group (PZ 0.0655) and the paclitaxel-plus-minocycline group (PZ0.0661). If we accept a P value of !0.10 as significant, then the three-drug group had significantly better survival than all other groups. 3.3. Toxicity The body weight of nude mice remained stable, and there were no significant differences in bodyweight changes between the groups (data not shown). The motor activity and the feeding behavior were normal. One mouse in the minocycline group died after 7 days of treatment. Another mouse in the manumycin-plus-paclitaxel group died on day 27 of drug administration.
Fig. 1. Effect of the combination of minocycline, manumycin, and paclitaxel on ARO xenograft growth. The mean tumor growth index was plotted for each treatment group. The error bars represented the 95% confidence intervals.
200
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Fig. 2. Effect of the combination of minocycline, manumycin, and paclitaxel on survival. Survival curves of treatment groups vs the control group. In each panel, the survival curves for the control group and a treatment group (as labeled) are shown. The horizontal axis represents the number of days since treatments began. The vertical axis represents the percentage of animals surviving.
Table 2 Survival outcomes with the various treatments Group
Median survival time (d)
95% Confidence internal
P value (vs control)
P value (vs manumycinC paclitaxelCminocycline group)
Control Manumycin Paclitaxel Minocycline ManumycinCpaclitaxel ManumycinCminocycline PaclitaxelCminocycline ManumycinCpaclitaxelCminocycline
23 28 28 23 28 22 33 46
17.29 25.31 22.34 19.27 22.34 20.24 24.42 44.48
1 0.4578 0.2888 0.7127 0.0521 0.9576 0.1204 0.001
0.001 0.0019 0.0035 0.004 0.0655 0.0127 0.0661 1
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201
4. Discussion
Acknowledgements
In this paper, we presented in vivo data from nude mice bearing ARO xenografts demonstrating that adding minocycline to the combination of manumycin and paclitaxel resulted in improved survival. At the 90% confidence level, the three-drug combination resulted in the better animal survival than any other possible combinations of the three drugs. Because each treatment group consisted of only eight animals, the power of the study was limited to the detection of large differences in any of the two-group comparisons. It has recently been suggested in the statistics literature that when only limited samples are available and a program of investigations is being undertaken, the traditional 0.05 level of significance may be relaxed and 0.10–0.20 (or higher) may be appropriate values for early-stage research [8,9]. P values between 0.05 and 0.20 were considered of interest for future investigations. Therefore, our hypothesis was supported by this experiment, and treatment with the triple-drug combination deserved further investigation in future. The tumor growth rate data were in agreement with the animal survival data. The triple combination group was the treatment group with the lowest mean tumor growth rate. Three-way ANOVA (generalized linear model analysis) demonstrated that there was a statistically significant interaction among the three drugs in their effect on the tumor growth rate. Minocycline is a safe and inexpensive drug. Its use as an antiangiogenic agent in combination with other chemotherapy for cancer has been investigated in preclinical animal models [10–12]. The combination of minocycline with a taxane and a farnesyltransferase inhibitor is novel. Although minocycline alone did not show any impressive antineoplastic effect, further exploration of its use in combination with other antineoplastic agents is warranted. In conclusion, the triple-drug combination of manumycin, paclitaxel, and minocycline was an effective combination against ATC, and resulted in improved survival in the nude mouse xenograft model. This study demonstrated the potential of a novel combination of a farnesyltransferase inhibitor, a taxane and an MMP inhibitor, which should be explored in future clinical trials.
This research was supported by a grant to SJY from Abbott Laboratories (Thyroid Research Advisory Council). The animal facility at The University of Texas M. D. Anderson Cancer Center is partially supported by the Cancer Center Support Grant (CA16672).
References [1] K.B. Ain, Anaplastic thyroid carcinoma: a therapeutic challenge, 16 (1999) 64–69. [2] S. Yeung, G. Xu, J. Pan, M. Christgen, A. Bamiagis, Manumycin enhances the cytotoxic effect of paclitaxel on anaplastic thyroid carcinoma cells, 60 (2000) 650–656. [3] G. Xu, J. Pan, C. Martin, C. Yeung, Angiogenesis inhibition in the in vivo antineoplastic effect of manumycin and paclitaxel against anaplastic thyroid carcinoma, 86 (2001) 1769–1777. [4] W.Z. Gu, S.K. Tahir, Y.C. Wang, H.C. Zhang, S.P. Cherian, S. O’Connor, J.A. Leal, S.H. Rosenberg, C. Ng, Effect of novel CAAX peptidomimetic farnesyltransferase inhibitor on angiogenesis in vitro and in vivo, 35 (1999) 1394–1401. [5] M.M. Feldkamp, N. Lau, A. Guha, Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects, 18 (1999) 7514–7526. [6] J.A. Fagin, K. Matsuo, A. Karmakar, D.L. Chen, S.H. Tang, P. Koeffler, High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas, 91 (1993) 179–184. [7] A. Yoshida, M. Fukazawa, H. Ushio, Y. Aiyoshi, S. Soeda, K. Ito, Study of cell kinetics in anaplastic thyroid carcinoma transplanted to nude mice, 41 (1989) 1–4. [8] R. Sposto, D.O. Stram, A strategic view of randomized trial design in low-incidence paediatric cancer, 18 (1999) 1183–1197. [9] N. Strauss, R. Simon, Investigating a sequence of randomized phase II trials to discover promising treatments, 14 (1995) 1479–1489. [10] E.A. Sotomayor, B.A. Teicher, G.N. Schwartz, S.A. Holden, K. Menon, T.S. Herman, E. Frei, 3rd, Minocycline in combination with chemotherapy or radiation therapy in vitro and in vivo, 30 (1992) 377–384. [11] B.A. Teicher, S.A. Holden, N.P. Dupuis, Y. Kakeji, M. Ikebe, Y. Emi, D. Goff, Potentiation of cytotoxic therapies by TNP470 and minocycline in mice bearing EMT-6 mammary carcinoma, 36 (1995) 227–236. [12] Y. Kakeji, A. Teicher, Preclinical studies of the combination of angiogenic inhibitors with cytotoxic agents, 15 (1997) 39–48.