Most cancer chemotherapeutic agents are administered at the maximum-tolerated dose (MTD) in short cycles with treatment breaks. However, MTD-based chemotherapies are often associated with significant toxicity and treatment breaks allow the opportunity for tumor regrowth and acquisition of chemoresistance. To minimize these drawbacks, a metronomic strategy, in which chemotherapeutics are administered at doses significantly below the MTD without treatment breaks, has been suggested by many investigators. The antitumor effect of metronomic chemotherapy may be partially due to inhibition of tumor angiogenesis, and it could be enhanced by a combination therapy, including antiangiogenic agents. In this study, we evaluated the synergistic effect of E10A, an adenovirus carrying the endostatin gene, the most potent inhibitors of tumor angiogenesis, in combination with weekly low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma. The E10A induced mRNA and protein expressions of endostatin in H891 cells in vitro. E10A significantly enhanced the in vivo tumor growth inhibitory effect of cisplatin. Immunohistochemical analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay and anti-CD31 antibodies revealed that the combination of E10A and cisplatin induced high levels of cell apoptosis and inhibited tumor angiogenesis. Importantly, E10A increased the platinum concentrations in tumors to fivefold higher than that induced by cisplatin alone.
Squamous-cell carcinoma of the head and neck accounts for 5% of newly diagnosed cancers in adults in the United States and 8% of cancers worldwide.1 Various strategies to improve outcomes by coordinating chemotherapy with surgery and radiotherapy have been tried, but the optimal schedule for integrating chemotherapy into the management of this disease has yet to be determined.2
For almost half a century, systemic therapy for cancer has been dominated by the use of cytotoxic chemotherapeutics. Most of these drugs are DNA-damaging agents or microtubule inhibitors that are designed to inhibit or kill rapidly dividing cells. They are often administered in single doses or short courses of therapy at the highest doses possible without causing life-threatening levels of toxicity, using what is termed the ‘maximum-tolerated dose’ (MTD). MTD therapy includes prolonged breaks (generally of 2–3 weeks in duration) between successive cycles of therapy. However, the progress that has been made in treating certain types of malignancy often comes at a high price, given the toxic side effects frequently associated with MTD-based chemotherapy.3 Shortly after the introduction of cytotoxic chemotherapy for the management of neoplastic diseases more than half a century ago, it became evident that the frequent excellent responses were mostly short-lived and that the relapsing tumors that initially responded to chemotherapy became drug resistant.
The historical emphasis on the concept that more drug is better has resulted in a relative neglect of other important parameters, such as the timing and duration of cytotoxic chemotherapy.4, 5 An alternative approach calls for the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks, termed metronomic chemotherapy,6 and does not target tumor cells directly, as does the MTD approach, but rather indirectly by inhibiting angiogenesis and vasculogenesis.3
Angiogenesis has an important role in the growth, progression and metastasis of tumors.7 Inhibiting the angiogenic process or targeting existing tumor vessels can be used for the treatment of tumors as an alternative or in parallel with conventional chemotherapy.8 Many antiangiogenic factors are under investigation and some are already being used in clinical practice with mixed results. Endostatin, a 20-kDa carboxyl-terminal fragment of type XVIII collagen, inhibits endothelial proliferation, angiogenesis and tumor growth.9, 10, 11 Endostatin induces endothelial cell apoptosis12 and confers survival advantage on cancer patients,13 but long-term systemic delivery of recombinant protein is expensive, painful experience, and may be insufficient to deliver high concentrations of the therapeutic molecule into the tumor.14 Antiangiogenic gene therapy can overcome these problems and represents a promising new approach for the treatment of cancer.
Cisplatin, or cis-diamminedichloroplatinum, an adduct of platinum (Pt), is one of the most potent anticancer drugs in clinical use and shows efficacy against several common types of solid tumors. The cytotoxic effect of cisplatin is thought to be due to its disruptive effect on DNA bases and the resultant DNA damage that induces apoptosis in cancer cells.15 It plays a major role in the treatment of a variety of cancers, including testicular, bladder, ovarian, head and neck, cervical, lung and colorectal cancer.16, 17 Combined treatments using cisplatin with other drugs have shown good results in several studies. In addition, several metronomic chemotherapy regimens with or without antiangiogenic agents are currently under investigation in both pre-clinical and clinical trials.3, 18 In this study, we evaluated the synergistic effect of E10A in conjunction with metronomic chemotherapy of weekly low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma.
Materials and methods
Human hypopharyngeal cancer cell line H891, which was established in the Yokohama City University School of Medicine, was used in this study.19 Cell line H891 was maintained in complete RPMI 1640 medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum.
The recombinant adenovirus vector
The recombinant adenovirus vectors carrying the human endostatin gene (E10A) and LacZ gene (Ad-LacZ) were generated as described previously.20, 21 The recombinant viruses contained the human endostatin following an IL-2-secretable signal, or LacZ genes under the control of the cytomegalovirus immediate-early promoter.22 All virus particles were amplified in 293 cells, purified by cesium chloride gradient centrifugation and titered using a standard plaque-forming assay. This study was approved by the Committee for Safe Handling of Living Modified Organisms of Kobe University and carried out according to the guidelines of the committee.
Detection of human endostatin gene expression on H891 cells infected with E10A
We infected H891 cells with E10A at multiplicities of infection (MOIs) of 0, 10, 25, 50 and 100 for 2 h, washed them once in phosphate-buffered saline (PBS) and incubated them at 37 °C in a fresh medium before harvesting at 24 h post-infection. The cells also were infected with E10A at an MOI of 25 and harvested at 24, 48 or 96 h post-infection. Total RNA was extracted from each group using Trizol (Invitrogen, Carlsbad, CA). The extracted mRNA was reverse transcribed using the Taq-Man Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA). Real-time quantitative polymerase chain reaction (PCR) using the Power SYBR Green PCR Master Mix (Applied Biosystems) was performed for the relative quantification of the mRNA expression according to a previously described method.23 The sequences for the primers were as follows: human endostatin—forward, 5-IndexTermTGGACAGGGAGGATTTTGAG-3, reverse, 5-IndexTermAGGCTGTGCCTTCCTACAGA-3; and β-actin—forward, 5′-IndexTermGGACTTCGAGCAAGAGATGG-3′, reverse, 5-IndexTermAGCACTGTGTTGGCGTACAG-3′. The PCR reactions were performed in the ABI7700 (Applied Biosystems) with PCR profiles as follows: 1 cycle for 5 min at 95 °C, 45 cycles for 30 s at 94 °C, 30 s at 56 °C, 1 min at 72 °C and with final cooling to 40 °C. The values of β-actin mRNA were used as an endogenous control to normalize for differences in the amount of total RNA.
Immunofluorescent detection of human endostatin protein expressions on H891 cells infected with E10A
In vitro H891 cells were infected with E10A at an MOI of 25. At 2 h post-infection, the medium was replaced with RPMI containing 10% fetal bovine serum. After a 72-h incubation, cells were collected and plated on slides. The slides were washed, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked for 20 min in Tris-buffered saline containing 10 ml l−1 goat serum. The cells were then incubated overnight with rabbit anti-human endostatin polyclonal antibody (Abcam, Cambridge, UK) diluted 1:50 in PBS. After washing three times, anti-rabbit secondary antibody-Alexa Fluor 488 (Dako, Carpinteria, CA) diluted 1:200 in PBS was added and the slides were incubated for an additional 30 min. The slides were then washed three times with PBS and the fluorescence signals were observed by a confocal laser scanning microscope (LSM 700; Carl Zeiss Meditec, Göttingen, Germany).
Treatment and measurements
The H891 cells (1 × 106) were suspended in 200 μl RPMI 1640 mixed with 200 μl BD Matrigel (Becton Dickinson, Franklin Lakes, NJ) and subcutaneously injected into the flanks of 5-week-old male nude mice (weight, 20–23 g) having a BALB/c (nu/nu) genetic background (CLEA Japan, Tokyo, Japan). When tumors with a 5- to 6-mm diameter had developed, 30 nude mice were randomly separated into five groups. Group 1 was given intratumoral E10A only (n=6), group 2 was given intraperitoneal cisplatin 2 mg kg−1 body weight, group 3 was given a combination of intratumoral E10A and intraperitoneal cisplatin (n=6), group 4 was given intratumoral Ad-LacZ and served as a control (n=6) and group 5 was given intraperitoneal PBS (n=6) and also served as a control.
The Ad-LacZ or E10A (1 × 109 plaque-forming units per 100 μl of PBS) were injected into the respective tumors on day 0. The tumor volume was measured two times a week until day 28 using the following formula: tumor volume (mm3)=a × b2 × 0.5236, where a is the longest diameter, b is the shortest diameter and 0.5236 is a constant to calculate the volume of an ellipsoid.
The mice were killed and the tumors were removed for histopathological analysis and measurement of Pt concentrations in the tumors at day 28 after initiation of the treatment.
The protocol was approved by the Kobe University Institutional Animal Care and Use Committee.
Determination of Pt concentrations
To measure Pt concentration in tumor samples, each weighed sample was completely reduced to ash by repeated treatment with nitric acid (for poisonous metal determination; Wako, Osaka, Japan), hydrogen peroxide (for atomic absorption spectrochemical analysis; Wako) and perchloric acid (for poisonous metal determination; Wako) under heat at 200 °C. Sample ashes were dissolved with 9 ml of 7% nitric acid and then analyzed by inductively coupled plasma-mass spectrometry using a Shimadzu ICPM-8500 (Shimadzu, Kyoto, Japan). We measured the Pt levels (m/z 195) in three representative samples. Contamination from tubes and other potential sources of Pt was carefully avoided. The concentration of Pt in each sample was calculated according to a linearly regressed curve prepared for Pt, using a standard solution for Pt (Wako). The values of a serially diluted standard solution showed linear regression with a line in the range from 1 to100 ng ml−1 for Pt.
Microvessel density analysis
For the immunohistochemical analyses, tumors were fixed in buffered formalin and embedded in paraffin. For detection of CD31 immunostaining, sections were probed with a mouse monoclonal CD31 (PECAM-1) antibody 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, followed by incubation with biotinylated polyclonal goat anti-mouse antibody 1:200 (Vector Laboratories, Peterborough, UK) and Vectastain Elite ABC Kit (Vector Laboratories). Positive reactions were visualized using 3,3-diaminobenzidine as a chromagen (DAB substrate kit; Vector Laboratories). Sections were counterstained with hematoxylin and mounted on glass coverslips. Images were captured using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) at × 200 magnification. The quantification of microvessel density (the maximum vascular area of the tumor) was assessed within the hot spot. The relative microvessel density among tumor cells was calculated by comparison with that of the control group being set to 1 as an arbitrary unit. To quantitate the apoptotic-positive cells per 8 random slides, we processed × 80 images using the Image J 1.41 software (National Institutes of Health, Bethesda, MD).
Apoptotic cells were identified using a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay (Takara Bio, Otsu, Japan) according to the manufacturer's instructions. Images were captured using the BIOREVO BZ-9000 microscope at × 200 magnification. The apoptotic cells were counted in five high-power fields in each slide in a blinded manner. The percentage of apoptotic cells among tumor cells was calculated as the apoptotic index, and this index was standardized by that of the control group set at 1 as an arbitrary unit. To quantitate the apoptotic-positive cells per 8 random slides, we processed × 80 images using the Image J 1.41 software.
The side effects of cisplatin include nephrotoxicity involving kidney damage, nausea and vomiting. We continuously monitored drug toxicity indexes such as weight loss and changes in behavior and feeding patterns throughout the treatment period.
To identify potential side effects in the treated mice, tissues of the liver, spleen and kidney were harvested and fixed in 4% neutral-buffered paraformaldehyde solution, embedded in paraffin, stained with hematoxylin and eosin and analyzed by pathologists in a blinded manner.
Determination of statistical significance was performed using a t-test for direct two-group comparisons and analysis of variance for multiple-group comparisons. All data are reported as the s.d. as an index of the variability of the original data points and reported as the mean±s.e. of three independent experiments if they were repeated. Statistical significance was set at P<0.05.
Endostatin gene and protein expression in H891 cells in vitro
To examine the level of endostatin gene expression in H891 cells infected with E10A, we quantified the mRNA expression of the endostatin gene in H891 cells infected with E10A at MOIs of 0, 10, 25, 50 and 100, respectively, by real-time PCR. As shown in Figures 1a and b, the human endostatin gene expression in H891 cells infected with E10A increased in proportion to the concentration of E10A (Figure 1a) and the time course at 25 MOI (Figure 1b).
At 3 days after transduction with E10A at 25 MOI, H891 cells were immune-stained with rabbit anti-human endostatin antibody. The immunohistochemistry clearly demonstrated that endostatin protein was expressed in the H891 cells infected with E10A. Extensive positive staining was observed in the cytoplasm of the E10A-infected cells (Figure 1d), but not in the non-E10A-infected control cells (Figure 1c). This indicated that E10A, an adenovirus carrying the human endostatin gene, may be a candidate for further investigation for local gene therapy in the human hypopharyngeal cancer cell line H891.
Inhibition of the growth of H891 xenograft tumors by combination of E10A and metronomic low-dose cisplatin
To investigate the combined effect of E10A and metronomic low-dose cisplatin, we established the H891 xenograft tumor model in athymic BALB/c (nu/nu) mice (5 weeks old). As shown in Figures 2a and b, after 4 weeks of treatment, the growth of tumors treated with the combined therapy was significantly more inhibited than that treated with either E10A only (P=0.01), cisplatin only (P=0.025), the Ad-LacZ control (P=0.02) or the PBS control (P<0.0001). The combined therapy significantly inhibited the growth of H891 xenografted tumors.
Impact of E10A on intratumoral cisplatin concentration in xenograft tumor model
To investigate the effect of E10A to increase the tumor concentration of cisplatin, we measured the concentration of Pt in tumor tissues in the combination (E10A+cisplatin), cisplatin-only and PBS control groups by inductively coupled plasma-mass spectrometry. As shown in Figure 3, we did not detect the Pt in the PBS control groups, but the combination of E10A and low-dose cisplatin significantly increased the intratumoral Pt concentration by about fivefold more than did the cisplatin-only treatment (P<0.0001).
Inhibition of angiogenesis by endothelial cell-specific CD31 analysis
To determine whether antitumor effect of E10A and metronomic low-dose cisplatin therapy was associated with suppression of angiogenesis, the status of vessel formation in treated H891 xenografts was assessed by immunohistochemistry with endothelial cell-specific CD31 (PECAM) staining. Figure 4 presents a representative result of the immunostaining. A high density of microvessels was present in the untreated H891 tumors, whereas E10A or metronomic low-dose cisplatin treatment alone was associated with a significant reduction in vascularization (Figure 4). With the combined treatment, a complete suppression of tumor angiogenesis was achieved compared with the E10A-only (P=0.031), cisplatin-only (P=0.001) and PBS control (P<0.0001) treatments.
Evaluation of tumor cell apoptosis by TUNEL assay
To investigate the synergistic effect of this combinational strategy, we evaluated the induction of sell apoptosis in tumors treated by each treatment group. Representative tumors harvested from each group were processed for apoptosis analyses. Apoptosis was evaluated by the TUNEL assay method. The apoptotic index of each group was expressed as its relative proportion to the untreated control. The number of staining-positive cells in the combined (E10A+cisplatin) group was significantly higher than that in the other groups (Figure 5b). Interestingly, the apoptosis index of E10A-only group was significantly higher than that of PBS group, but not that of Ad-LacZ group.
Absence of toxicity in all treatment groups
To investigate the clinical feasibility of this combination therapy with regard to the safety, we monitored weight loss and changes in behavior and feeding patterns and also performed the histopathological study of the main organs: liver, spleen and kidney. In this study, we did not find any significant differences of body weight among the all treatment groups, and no serious adverse effects, including cachexia, anorexia, behavior change or toxic death were observed in any treatment group. Furthermore, no pathological changes in the liver (Figures 6b, panel A), spleen (Figures 6b, panel B) or kidney (Figures 6b, panel C) were found by histopathological examination.
Chemotherapeutic agents can damage the DNA and disrupt DNA replication during cell proliferation. Cisplatin, or cis-diamminedichloroplatinum, an adduct of platinum, has long been used as an antineoplastic agent. When cisplatin was first approved for commercial use in 1978, the major reported toxicities were severe nausea and vomiting and a high incidence of renal dysfunction.24 At present, the conventional dosing schedule is designed to balance the toxicity and efficacy, but severe side effects and refractory treatment failures remain obstacles to the administration of this and most other chemotherapies. Thus, novel approaches are definitely required to achieve a high therapeutic response rate and to attenuate the severity of side effects.
Conventional dosing in chemotherapy calls for episodic application of a cytotoxic drug, and requires a period of rest during the therapy to allow normal cells to recover. With a low rate of replication and cell division (the proliferation index of endothelial cells in tumor vessels is usually <3%), the tumor-associated endothelial cells are only weakly damaged in the standard chemotherapy. Tumor-related angiogenesis can supply essential nutrients and oxygen for the remaining tumor cells, which makes tumor relapse possible.25 To minimize these drawbacks, a metronomic strategy, in which chemotherapeutics are administered at doses significantly below the MTD without treatment breaks, has been suggested by many investigators.3, 26, 27, 28 Another advantage of metronomic chemotherapy is the possibility of combining it with antiangiogenic drugs. One of the proposed benefits of combining metronomic chemotherapy with antiangiogenic therapies is a reduction of side effects and improved quality of life. As it is likely that chemotherapy will continue to be the mainstay of systemic cancer therapy for many years to come, designing more effective ways of administering and combining such drugs with the newest generation of molecularly targeted drugs will become important.
Although the antitumor activity of recombinant endostatin has been shown in a wide variety of experimental tumors, its overall therapeutic efficacy is generally moderate as shown in most in vivo studies. Endostatin gene therapy using E10A, a replication-deficient recombinant adenovirus containing a wild-type of the human endostatin gene, could directly express the highly bioactive protein in vivo by means of the eukaryotic expression system, and post-translational modification and long-term storage could overcome the need for the cumbersome daily administration of endostatin protein.29 The antiangiogenic effect of endostatin gene therapy with E10A, and the direct and indirect antitumor activity of metronomic low-dose cisplatin, offer an additive effect for tumor suppression. In this study, we combined intratumoral injections of E10A and intra-peritoneal injections of weekly low-dose cisplatin, and compared the antitumor and antiangiogenic effects of this combined therapy to several single-agent treatments in the H891. Our results demonstrated that the combined therapy significantly increased the tumor growth inhibitory effect compared to treatment with E10A alone or cisplatin alone (Figure 2). Consistent with our data, three recent studies showed that the combination of low, frequent-dose chemotherapy, plus an agent that specifically targets the endothelial cell compartment, controlled tumor growth much more effectively than did the cytotoxic agent alone.30, 31
Unlike dose-dense chemotherapy, the main targets of which are presumed to be proliferating tumor cells, the main targets of metronomic low-dose chemotherapy are the endothelial cells of the growing vasculature of the tumor.3 In this study, cisplatin or E10A alone could reduced the blood vessel density of tumors (Figures 4ai, iii and b); however, the combined treatment suppressed the tumor angiogenesis significantly more than did treatment with E10A or cisplatin alone (Figure 4). The aberrant structure and function of tumor neovessels results in a microenvironment within solid tumors that is characterized by elevated interstitial fluid pressure, inefficient tumor perfusion and significant areas of hypoxia and acidosis.32, 33 These factors contribute to poor intratumoral drug delivery and activity, and, in part, to the development of resistance to cytotoxic therapies.34 Thus, the inhibitory effect of tumor vascularization of the present combined therapy could improve the intratumoral drug delivery of cisplatin. It is worth noting that our plasma-mass spectrometry assay revealed that the combined therapy increased the intratumoral concentration of cisplatin to fivefold higher than that achieved by the cisplatin-only treatment (Figure 3).
The combination of E10A with cisplatin could strongly inhibit the tumor vascularization and consequently increase the intratumoral concentration of cisplatin. Furthermore, we found significantly more apoptotic cells in the combined treatment tumors than in those exposed to the other treatments (Figure 5). Clearly, the inhibition of tumor vascularization should decrease the supply of oxygen and nutrients to tumor cells, as well as decreasing the perfusion and vascular permeability,35 leading to apoptosis of the tumor cells. Further, each of these putative mechanisms would tend to reinforce the efficacy of cisplatin. The present combined approach of antiangiogenic gene therapy and metronomic low-dose chemotherapeutics is a promising strategy for markedly inhibiting tumor growth. A phase I clinical trial of E10A gene therapy has already been completed and the safety of this gene therapy has been confirmed.29 Further, this pre-clinical study of the combined therapy employing showed no serious general toxicity or specific toxicity evident via histopathological analysis of key organ tissues (Figure 6b) in this mouse model. These findings are encouraging for the clinical application of this combined therapy.
In conclusion, our results suggest that the combination of antiangiogenic gene therapy with metronomic low-dose chemotherapy is more effective in suppressing tumor growth in mice than either approach alone, with no evidence of obvious toxicity. The mechanism may variously involve the suppression of tumor angiogenesis, increased intratumoral concentration of chemotherapeutics and increased induction of apoptosis. The results of this study using the H891, HNSCC tumor model suggest that the combined therapy for the treatment of HNSCC warrants further clinical investigation.
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This research was supported by Japanese Research Project, Grant-in-aid for Scientific Research (B); project number 21390462 (TS), in part by Grants-in-Aid for Scientific Research; project number 20251283 and 21390462 (K-iN) and the Society for Promotion of International Oto-Rhino-Laryngology (SPIO), Japan (ZA).
The authors declare no conflict of interest.
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