Tripeptide tyroserleutide plus doxorubicin: therapeutic synergy and side effect attenuation
- Zhi-feng Zhu†1,
- Li-juan Chen†1,
- Rong Lu1, 2,
- Jing Jia1,
- Yu Liang3,
- Qiong Xu1,
- Chun-lei Zhou1,
- Li Wang1,
- Song Wang1 and
- Zhi Yao1, 4Email author
© Zhu et al; licensee BioMed Central Ltd. 2008
Received: 04 January 2008
Accepted: 25 November 2008
Published: 25 November 2008
Tripeptide tyroserleutide (YSL) is a novel small molecule anti-tumor polypeptide that has been shown to inhibit the growth of human liver cancer cells. In this study, we investigated the effects of YSL plus doxorubicin on the growth of human hepatocellular carcinoma BEL-7402 cells that had been transplanted into nude mice.
Nude mice bearing human hepatocellular carcinoma BEL-7402 tumors were treated with successive intraperitoneal injections of saline; low-, mid-, or high-dose doxorubicin; or low-, mid-, or high-dose doxorubicin plus YSL. Effects on the weight and volume of the tumors were evaluated.
Co-administration of YSL and high-dose doxorubicin (6 mg/kg every other day) prolonged the survival time of tumor-bearing mice as compared to high-dose doxorubicin alone. As well, the anti-tumor effects of mid- and low-dose doxorubicin (2 and 0.7 mg/kg every other day, respectively) were enhanced when supplemented with YSL; the tumor growth inhibition rates for YSL plus doxorubicin were greater than the inhibition rates for the same dosages of doxorubicin alone. The combination of YSL and doxorubicin decreased chemotherapy-associated weight loss, leukocyte depression, and heart, liver, and kidney damage as compared to doxorubicin alone.
The combination of YSL plus doxorubicin enhances the anti-tumor effect and reduces the side effects associated with doxorubicin chemotherapy.
According to the World Health Organization, 7.6 million people succumbed to cancer in 2005, accounting for 13% of all deaths. However, in China, the cancer death rate was 20.2%, and it is expected to reach 23.6% by 2023. Liver cancer is the third most common cause of death in China .
Anthracene-nucleus antibiotics are anti-tumor drugs that have been heavily researched and developed for the past twenty years. Among those, doxorubicin is the most commonly used and the most important; however, its use has been increasingly limited due to severe associated side effects, such as myocardial toxicity and bone marrow depression . Because the dosage of doxorubicin can be decreased when doxorubicin is given combination with other anti-tumor drugs, which can also elevate the therapeutic effect and relieve side effects of doxorubicin, combination therapy has become a common strategy for doxorubicin-based chemotherapy.
Tripeptide tyroserleutide (YSL) is a small molecule polypeptide that consists of three natural amino acids: L-tyrosine, L-serine, and L-leucine. In our laboratory, we have observed anti-tumor activity for YSL both in vitro and in vivo using the MTT method, the mouse model of ascites fluid-type hepatocarcinoma H22, and the nude mouse model of human hepatocellular carcinoma [3, 4]. In this study, we established a nude mouse model of human hepatocellular carcinoma in order to investigate the effect of YSL on tumor growth when given in combination with doxorubicin.
Human hepatocellular carcinoma BEL-7402 cells were purchased from Institute of Cell Biology, Shanghai, Academia Sinica. The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C. During cultivation, BEL-7402 cells remained free of mycoplasma or bacterial contamination.
Healthy female BALB/c (nu/nu) mice (4–5 weeks old, 18–22 g) were obtained from Chinese Academy of Medical Sciences (Beijing, China). The animals were housed at our university under specific pathogen-free conditions according to the guidelines of the Chinese Association for Laboratory Animal Care, using a laminar airflow rack. Animals had continuous access to sterilized food pellets and autoclaved water, and a 12-hr light/dark cycle. The temperature was maintained at 22–26°C and 50–70% relative humidity. All studies were performed according to the regulations of Tianjin Medical University experimental animal center and obtained the ethical approval from Tianjin Council for the Care of Animal in Medical Research.
Nude mouse model of human hepatocellular carcinoma 
To establish the human hepatocellular carcinoma model, BEL-7402 cells in exponential phase were grown to a density of 1 × 108/ml. 0.1 ml of the cell suspension was injected subcutaneously into the right back of every nude mouse. Mice with tumors of uniform volume were selected and randomly divided into different treatment groups when the tumor volume reached 100 mm3.
YSL plus high-dose doxorubicin combination therapy
YSL was obtained from Shenzhen Kangzhe Pharmaceutical, China. Doxorubicin (Adriamycin, ADM) was obtained from Zhejiang Haizheng Pharmaceutical Co., Ltd.
Nude mice bearing tumors were randomly divided into high-dose doxorubicin (6 mg/kg every other day), YSL (320 μg/kg/d), YSL (320 μg/kg/d) plus high-dose doxorubicin (6 mg/kg every other day), and saline (0.2 ml/d) groups; each group had twenty mice. All medicines were dissolved in 0.2 ml saline. Doxorubicin was administered intraperitoneally (IP) every other day, YSL and saline (control) were administered every day. For animals receiving combination therapy, the two medications were administered at a 4-hr interval. Mice received successive IP administrations for 30 days or until the death of mouse. Beginning on the day after the first administration, we observed activity, stool color and stool patterns of the mice in all treatment groups. We recorded the survival status of mice daily and calculated the rate of life extension according to the following formula: the rate of life extension = (average survival days of experiment group-average survival days of control group)/average survival days of control group × 100%.
YSL plus mid-dose doxorubicin combination therapy
Nude mice bearing tumors were randomly divided into mid-dose doxorubicin (2 mg/kg every other day), YSL (320 μg/kg/d), YSL (320 μg/kg/d) plus mid-dose doxorubicin (2 mg/kg every other day), and saline (0.2 ml/d) groups; each group had twelve mice. Mice received IP administrations for 30 successive days. The weights of mice were measured every three days until the experiment was terminated.
On the day after treatment termination, blood samples were obtained from the angular vein and anticoagulated with heparin. We measured hemoglobin levels, blood platelets count, and leukocyte counts. Hearts, livers, and kidneys were fixed immediately in 10% formalin. Paraffin-embedded sections of each tissue sample were stained with hematoxylin and eosin (HE). The weight and three perpendicular diameters of every tumor were recorded. Tumor volume was calculated by the following formula: V = (1/6) π ABC, where V is the tumor volume, and A, B, and C are the three tumor diameters. The rate of tumor inhibition = (average tumor weight of control group – average tumor weight of experiment group)/average tumor weight of control group × 100%.
YSL plus low-dose doxorubicin
Nude mice bearing tumors were randomly divided into low-dose doxorubicin (0.7 mg/kg every other day), YSL (320 μg/kg/d), YSL (320 μg/kg/d) plus low-dose doxorubicin (0.7 mg/kg every other day), and saline (0.2 ml/d) groups; each group had twelve mice. Treatments were administered IP for 60 successive days. Mouse characteristics including body weights, blood counts, and tumor volumes and weights were observed and recorded as described in the study of mid-dose doxorubicin.
Data are expressed as mean ± standard deviation (SD). Statistical significances of survival time differences were analyzed according to the Kaplan-Meier method using SPSS software. The statistical significance of other differences was tested using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. Statistical significance was set at P < 0.05.
YSL plus high-dose doxorubicin prolongs the survival time of nude mice bearing tumors compared with doxorubicin alone
After about 10 days of administration, the mice that received high-dose doxorubicin alone showed weight loss and decreased activity level. Similar changes were observed in the YSL plus doxorubicin group; however, in general, the condition of the combination group was better than that of the doxorubicin group. No obvious side effects were observed in YSL group.
Co-administration of YSL diminishes the side effects associated with mid-dose doxorubicin
The anti-tumor effects of YSL plus low-dose doxorubicin
Doxorubicin is a broad-spectrum antibiotic used widely for its anti-tumor activity. It is considered one of the most useful anti-tumor drugs available . However, it's clinical use is limited by its serious side effects including bone marrow depression , liver and kidney injury [8, 9], and cardiac toxicity . Cardiac toxicity is considered the main side effect; some consider doxorubicin's dose-dependent cardiac toxicity to be more dangerous than its other serious side effects . When doxorubicin is combined with other chemotherapeutic agents such as cyclophosphamide, bleomycin, and cisplatin, doxorubicin's cardiac toxicity is amplified. Therefore, doxorubicin dosage cannot be escalated and its use in combination therapy is limited. Identifying an acceptable therapeutic partner for doxorubicin remains an area of considerable clinical interest.
YSL is novel anti-tumor peptide that can inhibit the growth of human hepatocellular carcinoma cells without observable side effects. High performance and low toxicity are characteristic of YSL, and have become the basis efforts to use YSL and doxorubicin combination therapy to cure hepatoma. In this study, we imitated the clinical conditions of doxorubicin therapy and examined the therapeutic efficacy of YSL in combination with different dosages of doxorubicin. We evaluated the effects on the survival in a nude mouse model of human hepatocellular carcinoma.
Changes in tumor volume and weight are the most direct indices for judging the efficacy of anti-tumor therapy. We found that the tumor inhibition rate was enhanced when YSL was combined with mid- or low-dose doxorubicin, demonstrating that YSL enhances the anti-tumor effect of doxorubicin. In particular, the tumor inhibition rate of YSL plus low-dose doxorubicin was higher than that of the doxorubicin alone, whereas there was no statistically significant difference between the doxorubicin and saline groups. The manifestation of therapeutic action when low-dose doxorubicin is given in conjunction with YSL versus no therapeutic action when given alone suggests that YSL synergizes with doxorubicin. The inclusion of YSL can reduce the dosage required to obtain the clinical benefit of doxorubicin while limiting the potential side effects. These data are consistent with the principle of combined therapy for tumor treatment .
Other key indices for evaluating tumor therapy efficacy are survival time and survival quality. Research about survival quality has received increasing attention in the international community in recent years. Survival quality (quality-of-Life, QOL) has been widely used as an index to evaluate therapeutic results [13, 14]. At the 2005 meeting of the American Society of Clinical Oncology (ASCO), more than 98 articles about survival quality were presented. The completely new indexes-elevate survival quality and prolong survival time on tumor therapy curative effect was introduced clearly at ASCO 2007. In clinical anti-tumor therapy, elevating patient survival quality includes malignant tumor rehabilitation care , palliative treatment , and attenuation and synergy of radiotherapy and chemotherapy .
One focus of our research is on the attenuation and synergistic effects of YSL in combination chemotherapy, and the superiority of YSL for treating hepatoma. We first observed the attenuating effects of YSL in combination with deadly dosages of doxorubicin. High dosages of doxorubicin (6 mg/kg every other day) can induce death in mice; however, adding YSL to high-dose doxorubicin significantly prolongs the survival time of nude mice bearing tumors as compared to doxorubicin alone. These data suggest that YSL enhances nude mice's tolerance to doxorubicin, thereby increasing doxorubicin's clinical value. YSL in combination with mid-dose doxorubicin improves the survival status of nude mice bearing tumors and decreases doxorubicin-associated side effects such as weight loss, decreased body temperature, and reduced activity level. These data suggest that YSL plus doxorubicin both prolongs the survival time of nude mice bearing tumor and enhances their survival quality, which has important clinical implications.
Bone marrow depression is a common side effect of doxorubicin, and the influence on leukocyte counts is the most obvious. About 60–80% of patients have leukocyte depression after 10–15 days of doxorubicin therapy, and they recover by about day 21 of therapy . YSL has strong protective effect on doxorubicin-induced leukocyte depression. In this study, YSL given in combination with mid-dose doxorubicin increased the number of leukocytes; thus, the inclusion of YSL in doxorubicin treatment regimens could reduce rate of infectious complications.
During therapy, doxorubicin can induce fatal cardiac toxicity, and doxorubicin-induced liver and kidney injury can be life threatening. Mid-dose doxorubicin can injure the heart, liver, and kidneys to varying degrees. We found that the degree of injury was reduced in the YSL combination group as compared to the doxorubicin group; that is, YSL seemed to prevent or repair doxorubicin-induced heart, liver, and kidney injury. The cardio- and hepatoprotective effects of YSL were especially obvious. In the YSL combination group the structure of cardiocyte was clear, hepatic lobules was complete, and the structure of the kidney cortical labyrinth and medullary ray was clear compared to the doxorubicin group.
The elevated doxorubicin levels in the blood conduce to reaching a high concentration in tumor tissue to increase the anti-tumor effects of doxorubicin . However, some organs with abundant blood supply such as heart, liver and kidney will be damaged because of high doxorubicin concentration. In this study, we found that high- and mid-dose doxorubicin had obvious anti-tumor effects on human hepatocellular carcinoma BEL-7402 cells, but doxorubicin also influenced the common status and basic vital signs of nude mice bearing tumors severely. Doxorubicin's side effects include hypo-leukocytosis, cardiac injury, liver and kidney toxicity, weight loss, and survival time decurtation. Compared with control group, low-dose doxorubicin induced less severe side effects, but had no significant inhibition on the growth of human hepatocellular carcinoma BEL-7402 cells in nude mice. Combination therapy with YSL significantly protected the mice from doxorubicin-induced side effects and increased the anti-tumor effects of doxorubicin. YSL could induce the apoptosis of human hepatocellular carcinoma BEL-7402 cells . And Xu et al found that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) could significantly increase the anti-tumor effects of doxorubicin through inducing the apoptosis of cancer cells . So we think that the mechanisms enabling YSL to augment the cytotoxicity of doxorubicin may correlate well with the increased ability of doxorubicin when combined with YSL to induce apoptosis. Also YSL significantly increased the expression of PTEN in human hepatocellular carcinoma BEL-7402 cell. Over-expression of PTEN could increase the sensitivity to doxorubicin in human breast cancer MCF-7 cells . The boosting of activity of doxorubicin when combined with YSL may result from the increased expression of PTEN to increase the sensitivity of BEL-7402 cells to doxorubicin.
In addition, our study observed the effects of co-administration of YSL and high-, mid-, and low-dose doxorubicin on nude mice bearing tumors. Although few side effects were recorded in low-dose doxorubicin group, there was no significant difference in tumor volumes and weights between the low-dose doxorubicin group and saline group. The tumor inhibition rate of YSL combination group was higher than that of the low-dose doxorubicin group, significantly different as compared to the saline group. YSL might enhance the efficacy of doxorubicin and have a dosage sparing effect which can reduce the dosage of doxorubicin to reduce its side-effects. Hence, it is possible that YSL can act as a new anti-tumor drug for the clinical development of combination with doxorubicin. Also YSL could reduce doxorubicin-induced side effects in combination with mid- and high-dose doxorubicin which produced significant anti-tumor effects. For example YSL prolonged the survival time of nude mice bearing tumor and overcame the negative side effects of doxorubicin by protecting hematological and vital organ health. Doxorubicin can undergo redox cycling to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the side effects of the drug . Further research needs to be carried out to test whether YSL would interfere with oxidant-generating activity of doxorubicin to inhibit side effects of drug. Moreover, the mechanism accounting for side-effects of doxorubicin mainly depends on its cytotoxicity. Further experiment is required to show that YSL could selectively induce apoptosis of human hepatocellular carcinoma cells but not normal cells from various organs.
In this study, the potential attenuation and synergistic effect of tripeptide tyroserleutide plus doxorubicin against human hepatocellular carcinoma in nude mice were investigated. Co-administration of YSL and low-, mid-, or high-dose doxorubicin respectively decreased chemotherapy-associated weight loss, leukocyte depression, and heart, liver, and kidney damage as compared to doxorubicin alone. Although there were no statistically significant differences in tumor weights between doxorubicin alone group and YSL plus doxorubicin group, YSL plus doxorubicin produced greater tumor inhibition rate than doxorubicin. The combination of YSL plus doxorubicin increased the sensitivity of tumors to chemotherapeutic drugs and reduces the side effects associated with doxorubicin chemotherapy.
Doxorubicin or Adriamycin
hematoxylin and eosin
analysis of variance
American Society of Clinical Oncology
tumor necrosis factor-related apoptosis inducing ligand
white blood cells
This work was supported by Grants for the National High Technology Research and Development Program of China ("863" Program, China) (2004AA2Z3170, 2005AA2Z3D40) and the National Basic Research Program (973 Program, China) and by an important project grant (03007) from the Department of Education of China.
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