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Aug 20, 2009 - Combination chemotherapy of doxorubicin and paclitaxel for hepatocellular carcinoma in vitro and in vivo. Cheng Jin · Haimin Li · Yong He ...
J Cancer Res Clin Oncol (2010) 136:267–274 DOI 10.1007/s00432-009-0658-5

ORIGINAL PAPER

Combination chemotherapy of doxorubicin and paclitaxel for hepatocellular carcinoma in vitro and in vivo Cheng Jin · Haimin Li · Yong He · Min He · Ling Bai · Yunxin Cao · Wenjie Song · Kefeng Dou

Received: 12 May 2009 / Accepted: 3 August 2009 / Published online: 20 August 2009 © Springer-Verlag 2009

Abstract Purpose Systemic combination chemotherapy is the only option for patients with unresectable hepatocellular carcinoma (HCC) not suitable for intra-arterial treatment. However, no systemic chemotherapy has been able to provide durable remission. The search for a new combination of drugs for HCC is signiWcant. The combination of doxorubicin and paclitaxel shows promise in breast cancer therapy. This study was carried out to determine the synergistic eVect of combined doxorubicin and paclitaxel in the two HCC cell lines: HepG2 and Huh7 in vitro and murine HCC H22-bearing BALB/c mice in vivo. Methods The morphology of the two cell lines treated with drugs was photomicrographed. The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay was used to determine the number of surviving cells. Cell cycle was evaluated by Xow cytometry. Cell viability was measured

by the ability of single cells to form colonies in vitro. Antitumor activities against subcutaneoulsy implanted solid tumor induced by H22 cells in mice were evaluated. Results Our data demonstrated that the cytotoxicity produced by doxorubicin and paclitaxel was additive in HCC cells, while it was mainly held in the G2/M phase of the cell cycle by paclitaxel. In vivo anti-tumor activity assay also showed that the combination of the two drugs resulted in more signiWcant tumor regression, compared to the single one. Conclusion The study may provide a new combination of cytotoxic drugs for HCC chemotherapy. Keywords Hepatocellular carcinoma · Doxorubicin · Paclitaxel · Combination chemotherapy

Introduction C. Jin, H. Li, Y. He, M. He and L. Bai contributed equally to this work. C. Jin · H. Li · Y. He · W. Song · K. Dou (&) Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected] M. He Department of Surgery, Qinshui Prefectural People’s Hospital, Jincheng 048200, China L. Bai Department of Clinical Laboratories, Xi’an Gaoxin Hospital, Xi’an 710075, China Y. Cao Department of Immunology, Fourth Military Medical University, Xi’an 710032, China

Hepatocellular carcinoma (HCC) is currently the Wfth most common solid tumor worldwide, and the fourth leading cause of cancer-related death (World Health Organization, 2008; Parkin et al. 2005). It is responsible for 662,000 deaths per year, as the annual incidence roughly equals the annual mortality (Llovet et al. 2003; Thorgeirsson and Grisham 2002). Surgical resection is the only hope for long-term survival of HCC patients. However, more than 80% of patients present with advanced or unresectable disease, and for those patients who do undergo resection, the recurrence rates can be as high as 50% at 2 years (Nagasue et al. 1993; Yamamoto et al. 2005; Tanaka et al. 2005). Intensive research eVorts have been directed toward the identiWcation of novel treatment strategies for HCC. Many patients seek systemic therapy. HCC is only moderately sensitive to systemically administered single agents.

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Doxorubicin, a member of the anthracycline ring antibiotics with a broad spectrum of antitumor activity, is commonly used, but the objective response rates from 13 published trials were < 20%, and the median survival was only 4 months (Nerenstone et al. 1988). Because of the low response rate of a single agent, systemic combination chemotherapy is usually used to solicit synergism between the treatment drugs and to induce a better tumor response. The combination of doxorubicin and cisplatin by the intrahepatic-arterial route was more active (response rate = 56%) (Carr et al. 1993). A pivotal phase III trial of doxorubicin combination chemotherapy (cisplatinum, interferon, doxorubicin and 5-Xuorouracil, PIAF) showed a statistically signiWcant diVerence in response rate favoring PIAF (Yeo et al. 2005). However, the other combinations of drugs have not provided clinical beneWt or prolonged survival for patients with advanced HCC (Johnson 2002, 2003; Palmer et al. 2004). Therefore, a search for a new combination of drugs that can beneWcially interact with each other is important to improve the therapeutic eYcacy in the management of HCC. Paclitaxel is one of the best anti-neoplastic drugs found in the past decades. It has been clinically used in the treatment of various cancers, especially breast and ovarian cancers (Adams et al. 1993). Paclitaxel has a unique mechanism of action. It promotes the polymerization of tubulin, unlike other microtubule agents such as vinca alkaloids, which induce the disassembly of microtubules. The microtubules formed in the presence of paclitaxel are extraordinarily stable and dysfunctional, thereby causing the death of the cell by disrupting the normal tubule dynamics required for mitotic cell division. Geng reported that docetaxel inhibited the growth of human HCC SMMC-7721 cells and induced apoptosis (Geng et al. 2003). Okano indicated the growth inhibition of liver cancer cells by paclitaxel and the involvement of extracellular signal-regulated kinase and apoptosis (Okano et al. 2007). Zhang reported that paclitaxel had signiWcant growth inhibitory eVect on angiogenesis and metastasis of human HCC in nude mice (Zhang et al. 2005). Iesalnieks indicated that paclitaxel promoted liver graft survival in rats and inhibited HCC growth Fig. 1 The molecular formulas of doxorubicin and paclitaxel

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J Cancer Res Clin Oncol (2010) 136:267–274

in vitro and was a potentially useful drug for transplant patients with liver cancer (Iesalnieks et al. 2002). Strumberg reported that paclitaxel might have activity for HCC in a phase I study (Strumberg et al. 1998). As doxorubicin and paclitaxel have synergistic activity in breast cancer therapy (Shi et al. 2008; Piccart-Gebhart et al. 2008; Hong et al. 2008; Bourgeois et al. 2006), it may be possible to combine the two drugs for chemotherapy of HCC. This study was carried out to determine whether the cytotoxic properties of the two drugs were additive when administered together at clinically relevant concentrations into the two human HCC cell lines: HepG2 and Huh7 in vitro and murine HCC H22-bearing BALB/c mice in vivo.

Materials and methods Materials Drugs Doxorubicin was purchased from Beijing HuaFeng United Technology (Beijing City, China). Paclitaxel injection (H20063662, 30 mg/5 ml) was from Beijing Sihuan Pharmaceutical Co. Ltd (Beijing City, China). The molecular formulas of doxorubicin and paclitaxel are shown in Fig. 1. Doxorubicin was dissolved in phosphate buVered saline (PBS) medium (pH 7.4) at a concentration of 50 g/ml for the actual test. A paclitaxel stock solution of 10 g/ml prepared in PBS was kept at ¡20°C and thawed for use. In the preliminary experiment, 50% inhibiting concentration (IC50) doses of doxorubicin for HepG2 and Huh7 cells were 0.87 and 0.79 g/ml, respectively. IC50 doses of paclitaxel for HepG2 and Huh7 cells were 8.52 and 16.31 ng/ml, respectively. According to the results, cells were exposed to concentrations of doxorubicin at 0.5 g/ml and/or paclitaxel at 10 ng/ml for 6 h. After treatment, the medium with the drugs was absorbed and then the fresh Dulbecco’s modiWed Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) was added.

J Cancer Res Clin Oncol (2010) 136:267–274

Cells culture The HepG2 and Huh7 cells were kindly provided by MD. SR Ma (Department of Pathology, Xijing Hospital, Fourth Military Medical University) and MD. SD Chen (Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University), respectively. The H22 cells were purchased from the Experimental Animal Center of Fourth Military Medical University (Xi’an city, China). The cells were cultured as monolayers in DMEM supplemented with 10% FBS, penicillin and streptomycin in a humidiWed atmosphere of 95% air and 5% CO2 at 37°C, and subcultured twice weekly. For experiments, the cells were grown in glass culture Xasks and used when in the exponential growth phase. Animals Male BALB/c mice (20 § 2 g) were supplied by the Experimental Animal Center of Fourth Military Medical University (Xi’an city, China). The animal were acclimatized at a temperature of 25 § 2°C and a relative humidity of 70 § 5% under natural light/dark conditions for 1 week before dosing. The study protocol was approved by and performed in accordance with the Committee of the Use of Live Animals in Teaching and Research at the Fourth Military Medical University. Methods Cell morphology The HepG2 and Huh7 cells were plated at 1 £ 105 cells per well in six-well tissue culture plates for 24 h. Then administration of drugs was conducted as previously described in the Sect. ”Drugs”. Then, phase-contrast photomicrography was carried out using a Nikon TE2000-S microscope (Japan). MTT assay The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to determine the number of surviving cells. The HepG2 and Huh7 cells were plated at 500 cells per well in 96-well tissue culture plates and allowed to attach for 24 h. Administration of drugs was conducted as previously described in the Sect. ” Drugs”. Six hours after administration, 20 l of MTT (5 mg/ml) was added to each well and the plates returned to the incubator for 4 h. At this point, special care was taken when removing untransformed MTT by careful aspiration so as not to disturb the blue formazan crystals adhering to the cells. Then 150 l DMSO was added to each well to dissolve the formazan crystals while slightly agitating the cells on an

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automated shaker. Thereafter, the absorbance of the suspension was measured at 490 nm on an ELISA reader. The amount of formazan present is proportional to the number of viable cells, as only living cells will reduce MTT to blue formazan. Results were expressed as a percentage of the absorbance present in untreated cells compared to that in the treated cells. Flow cytometry study The HepG2 and Huh7 cells were plated at 1 £ 106 cells per well in six-well tissue culture plates and allowed to attach for 24 h. Administration of drugs was conducted as previously described in the Sect. ”Drugs”. Six hours after administration, the cells were Wxed according to the unary-color staining procedure. BrieXy, the cells were mechanically removed from the tissue culture plates and centrifuged at 1,000 rpm for 5 min. The supernatant was aspirated and the pellet was resuspended in 1 ml of PBS. The suspension was mixed continually while 2 ml of cold ethanol was added. The cells were stored at 4°C. Before Xow cytometric analysis, cells were stained with propidium iodide (PI) as Xuorescent marker containing 3 Kunitz RNase. Flow cytometry analysis was performed using an ELITE ESP machine (Beckman-Coulter, USA). Cell survival assay Cell viability was measured by the ability of single cells to form colonies in vitro. The HepG2 and Huh7 cells were plated at 500 cells per well in six-well tissue culture plates and allowed to attach for 24 h. The administration of drugs was conducted as previously described in the Sect. ”Drugs”. Six hours after administration, the medium was removed by aspiration and fresh DMEM supplemented with 10% FBS was added. Then, the cells were allowed to grow under standard culture condition for 10–14 days. After this time interval, macroscopic colonies were stained with Giemsa and were counted manually. In vivo anti-tumor activity In vivo anticancer activity was evaluated against H22-bearing BALB/c mice (n = 6). Mice were inoculated subcutaneously into the back with H22 cells. After 10 days, doxorubicin at 1 mg/kg per day and/or paclitaxel at 2 mg/ kg per day was given by intravenous injection each third day (six times) for 15 days. The mice were treated with PBS as the control. Tumor volumes [(major axis) £ (minor axis)2 £ 1/2] were measured at deWned time periods. The inhibition rate of tumor (%) was calculated using the following formula: (tumor volume of control - tumor volume of experiment)/tumor volume of control £ 100%. Pieces of

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tumor tissue were retrieved and Wxed in buVer neutral formalin for histological examination. Statistical analysis All the experiments were repeated three times. All the data are presented as mean § SD. The signiWcance of diVerences between the groups was determined with one-way ANOVA (SPSS10.0 statistical software), with the level of signiWcance set at P < 0.05.

Results Cell morphology

J Cancer Res Clin Oncol (2010) 136:267–274

presented in Table 1. The presence of the drugs alone or in combination reduced the values of HepG2 and Huh7 cells. HepG2 cells were more sensitive to paclitaxel than Huh7 cells. There was no diVerence in the cytotoxicity of doxorubicin between the two cell lines. The signiWcant synergistic cytotoxic eVect of the two drugs was observed on the two tumor cell lines, compared to the single drug. Percentage of cells in the G2/M phase After administration of paclitaxel, the cells were mainly blocked in the G2/M phase. For doxorubicin, there was no signiWcant statistical diVerence between the phases. A combination of the two drugs also resulted in HepG2 cells being blocked in the G2/M phase (Table 2).

The morphological consequences of 6 h doxorubicin and/or paclitaxel exposures on the HepG2 and Huh7 cells are shown in Figs. 2 and 3. After administration of drugs, there was a clear change in cellular morphology, with cells being unable to spread. Compared with control (without drugs), the part of the two HCC cell lines treated with drugs were impaired and dead. The eVect of combination of doxorubicin and paclitaxel was more signiWcant than that of a single drug.

Cell survival assay

MTT assay

In vivo anti-tumor activity

The eVects of doxorubicin and paclitaxel on the survival rate of the two HCC cell lines 6 h after administration are

To assess the anti-tumor activity in vivo, doxorubicin and/ or paclitaxel were injected through the tail vein at clinically

Fig. 2 Phase-contrast photomicrographs of HepG2 cells following a 6 h treatment with blank (a) doxorubicin (b), paclitaxel (c) and doxorubicin + paclitaxel (d), respectively

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Plating eYciency was 71 and 55% for HepG2 and Huh7 cells, respectively. Figure 4 indicates that the surviving fractions of the two cell lines were aVected by doxorubicin and/ or paclitaxel and the two drugs had synergetic cytotoxic eVect. The cytotoxicity of doxorubicin was much higher than that of paclitaxel for both HepG2 and Huh7 cells.

J Cancer Res Clin Oncol (2010) 136:267–274

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Fig. 3 Phase-contrast photomicrographs of Huh7 cells following a 6 h treatment with blank (a) doxorubicin (b), paclitaxel (c) and doxorubicin + paclitaxel (d), respectively

Table 1 The cytotoxic eVects of doxorubicin and/or paclitaxel on HepG2 and Huh7 cells Cell

HepG2 Huh7 a

Concentration of doxorubicin (g/ml)

Concentration of paclitaxel (ng/ml)

0



0

10 45.33 § 9.18 a

0.5

73.46 § 8.13

34.67 § 3.84

0



81.63 § 7.81a

0.5

70.98 § 7.60

62.72 § 7.48

Discussion

P < 0.05 versus combination of drugs

Table 2 Cell cycle distribution of HepG2 and Huh7 cells incubated with doxorubicin and/or paclitaxel Cells

Treatment

G1 (%)

HepG2 Control 57.1 § 3.8 (without drugs)

Huh7

a b

S (%)

G2/M (%)

33.2 § 2.6

9.7 § 1.6

45.1 § 0.6

16.9 § 3.1

Doxorubicin

38.0 § 3.7

Paclitaxel

41.0 § 12.0 25.5 § 11.8 33.5 § 0.2a

Doxorubicin + paclitaxel

37.0 § 7.8

27.0 § 26.6 36.0 § 18.8a

Control 51.9 § 0.1 (without drugs)

43.7 § 2.5

4.4 § 2.5

Doxorubicin

50.1 § 2.9

45.1 § 1.9

4.8 § 0.9

Paclitaxel

58.5 § 12.2 26.9 § 21.2 14.6 § 9.1a,b

Doxorubicin + paclitaxel

44.5 § 2.3

P < 0.05 versus control without drug P < 0.05 versus combination of drugs

relevant doses to H22-bearing mice. As shown in Fig. 5, tumor regression was observed for mice treated with the drugs. The tumor burden of doxorubicin + paclitaxeltreated mice was much smaller compared with single or non-drug-treated mice. Three days after the Wnal administration, the inhibition rates of tumor (%) were 46.9, 34.3 and 67.4% for doxorubicin, paclitaxel and doxorubicin + paclitaxel, respectively.

50.4 § 6.3

5.1 § 4.0

Although surgery oVers the only hope of cure for HCC, patients with inoperable or metastatic disease have a dismal prognosis. For disease conWned to the liver, various locoregional treatments may oVer useful palliation. These include intra-arterial infusion of combination chemotherapy (Carr et al. 1993; Patt et al. 1994), chemoembolization (Pelletier et al. 1990; Groupe d’Etude et de Traitement du Carcinome Hepatocellulaire 1995) and selective internal radiation treatment (Lau et al. 1998). However, it is only possible in selected cases, and delivery requires specialized facilities. Patients with extrahepatic metastasis and blocked portal venous system are usually not candidates for intra-arterial treatment, and systemic chemotherapy is the only remaining option. Single-agent chemotherapy has met with only very limited success. Response rates for single agent chemotherapy, usually doxorubicin, are approximately 15–20%. To improve the eYcacy of systemic chemotherapy, combination chemotherapy is widely

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272 Fig. 4 The HepG2 and Huh7 cells, incubated without drugs (a, e), with doxorubicin (b, f), paclitaxel (c, g) and doxorubicin + paclitaxel (d, h), were stained with Giemsa, photographed and analyzed for their proliferation eYciency (above). Cell surviving fractions for HepG2 and Huh7 cells treated with doxorubicin, paclitaxel and doxorubicin + paclitaxel, respectively (below). (*P < 0.05 vs. single drug)

J Cancer Res Clin Oncol (2010) 136:267–274

a

b

c

d

e

f

g

h

HepG2

Huh7

0.7

Surviving Fraction

0.6 0.5

Doxorubicin

0.4

Paclitaxel

0.3

Doxorubicin+Paclitaxel

0.2

*

0.1 0

*

HepG2

Huh7

Cells

a

d Relative tumor volume

Fig. 5 Anti-tumor eVects (in terms of tumor growth) of doxorubicin, paclitaxel and doxorubicin + paclitaxel on H22-bearing mice. a The mice with transplanted H22 tumor. Tumor nodule was indicated by black arrow. b Tumor was retrieved. c Tumor tissue H&E stain. d In vivo anti-tumor activity assay

c

b

8 7 6 5

Control

4

Doxorubicin Paclitaxel

3

Doxorubicin+Paclitaxel

2 1 0

0

5

10

15

20

25

30

35

Time (Days)

considered and used in oncology practice. It appears to give a higher response rate (20–30%), although remissions are usually short and survival advantage has not been convincingly demonstrated (Leung and Johnson 2001).

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Moreover, many regimens have not provided evidence of eYcacy for HCC patients. Therefore, there is an urgent need for new and eVective treatment regimens in the management of HCC.

J Cancer Res Clin Oncol (2010) 136:267–274

The outcomes may be improved by the combination of doxorubicin and the promising new taxanes, such as paclitaxel. Preclinical and clinical studies have shown that paclitaxel is active against HCC in cell lines, xenografts and patients. Doxorubicin and paclitaxel have synergistic cytotoxicity in breast cancer and their combination was used clinically. We therefore investigated the possibility of combining doxorubicin and paclitaxel to achieve additive activity in overcoming HCC. In the study, the morphology of HepG2 and Huh7 cells markedly changed after treatment with doxorubicin and/or paclitaxel. The partial tumor cells were impaired and dead. This may be mainly due to the cytotoxicity of the drugs. The combination of the two drugs showed more signiWcant eVect on cellular morphology. This suggested that the two drugs had synergistic eVect on HCC cell lines. The MTT assay was used to demonstrate the cytotoxicity of the applied drugs on the HCC cells. Our results indicated that the cytotoxic eVect of doxorubicin and/or paclitaxel was signiWcant for both HepG2 and Huh7 cells. The combination of drugs resulted in preliminary synergistic cytotoxicity for the two HCC cell lines, compared to the single drug. The cytotoxic eVect of doxorubicin and/or paclitaxel was expressed by the ability of the drugs to block the tumor cells in the G2/M phase of the cell cycle. With the addition of paclitaxel to the two HCC cells culture, the number of cells in the G2/M phase increased. However, no signiWcant eVect of doxorubicin on tumor cell cycle was observed. This suggested that the change in the cell cycle was drugdependent. The synergistic eVect of these two drugs was less obvious after administration. To understand further the synergistic cytotoxicity of the two drugs, cell survival assay was performed. Clearly, the treatment with doxorubicin and/or paclitaxel resulted in an enhancement in the fraction of cell becoming clonogenically incompetent. The cytotoxic eVect of doxorubicin was more signiWcant than that of paclitaxel at these administered doses. Synergistic cytotoxicity of these two drugs on HepG2 and Huh7 cells was observed. This was consistent with the cell morphology and the MTT assay. The results demonstrated that the therapeutic eYciency of the two drugs was additive for HCC cell lines. So the co-administration of the two drugs may result in a beneWcial gain in HCC chemotherapy. To provide evidence on the true eYcacy of this combination, the anti-tumor activity in vivo was studied using H22bearing mice. The data demonstrated that the combination of doxorubicin and paclitaxel had a synergistic eVect, causing more signiWcant tumor regression. However, complete tumor regression was not observed. This suggested that the dose of administration may be modiWed to produce a better anti-tumor eVect in future studies.

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Shi reported that the paclitaxel–doxorubicin sequence was more eVective in breast cancer cells with heat shock protein 27 overexpression (Shi et al. 2008). Piccart-Gebhart indicated that taxanes alone or in combination with anthracyclines may be used as Wrst-line therapy of patients with metastatic breast cancer (Piccart-Gebhart et al. 2008). Hong indicated the use of a combination of pegylated liposomal doxorubicin and paclitaxel as salvage chemotherapy in metastatic breast cancer previously treated with anthracycline (Hong et al. 2008). Bourgeois reported the use of pegylated liposomal doxorubicin combined with weekly paclitaxel as Wrst-line treatment in patients with metastatic breast cancer in Phase I–II study (Bourgeois et al. 2006). These studies demonstrated the eYcacy of combination chemotherapy of the two drugs and suggested the possibility of the combination for HCC in vivo. But to date, there is no report of experimental evidence that the cytotoxic eVects of a combination of doxorubicin and paclitaxel are additive for HCC. This study has proved that the combination resulted in synergistic anti-tumor activity in vitro and in vivo. Therefore, based on our Wndings, further studies are needed to provide data for clinical application.

Conclusions Paclitaxel showed the ability to block the tumor cells in the G2/M phase of the cell cycle. The cytotoxicity produced by doxorubicin and paclitaxel was additive in vitro and in vivo when they were administered together at clinically relevant concentration. This study may provide a new combination of drugs for HCC chemotherapy and further clinical studies are signiWcant and necessary. Acknowledgments This work was supported by a grant from the National Nature Science Foundations of China: No30571828. The authors would like to thank Prof. F.Q. Zhang (Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University) for the technical assistance.

References Groupe d’Etude et de Traitement du Carcinome Hepatocellulaire (1995) A comparison of lipiodol chemoembolization and conservative treatment for unresectable hepatocellular carcinoma. N Engl J Med 332:1256–1261 Adams JD, Flora KP, Goldspiel BR, Wilson JW, Arbuck SG, Finley R (1993) Taxol: a history of pharmaceutical development and current pharmaceutical concerns. J Natl Cancer Inst Monogr 5:141–147 Bourgeois H, Ferru A, Lortholary A, Delozier T, Boisdron-Celle M, Abadie-Lacourtoisie S et al (2006) Phase I–II study of pegylated liposomal doxorubicin combined with weekly paclitaxel as Wrstline treatment in patients with metastatic breast cancer. Am J Clin Oncol 29:267–275 Carr BI, Iwatsuki S, Baron R, Jain A, Selby R, Madariaga J et al (1993) Intrahepatic arterial cisplatinum and doxorubicin with or without

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274 lipiodol for advanced hepatocellular carcinoma (HCC): a prospective randomized study. Proc Annu Meet Am Soc Clin Oncol 12:A668 Geng CX, Zeng ZC, Wang JY (2003) Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis. World J Gastroenterol 9:696–700 Hong RL, Lin CH, Chao TY, Kao WY, Wang CH, Hsieh RK et al (2008) A phase-I study evaluating the combination of pegylated liposomal doxorubicin and paclitaxel as salvage chemotherapy in metastatic breast cancer previously treated with anthracycline. Cancer Chemother Pharmacol 61:847–853 Iesalnieks I, Tange S, Scherer MN, Graeb C, Frank E, Jauch KW et al (2002) Paclitaxel promotes liver graft survival in rats and inhibits hepatocellular carcinoma growth in vitro and is a potentially useful drug for transplant patients with liver cancer. Transplant Proc 34:2316–2317 Johnson PJ (2002) Hepatocellular carcinoma: is current therapy really altering outcome? Gut 51:459–462 Johnson PJ (2003) Are there indications for chemotherapy in hepatocellular carcinoma? Surg Oncol Clin N Am 12:127–134 Lau WY, Ho S, Leung WT, Chan M, Ho R, Johnson PJ et al (1998) Selective internal radiation therapy for non-resectable hepatocellular carcinoma with intra-arterial infusion of yttrium-90 microspheres. Int J Radiat Oncol Biol Phys 40:583–592 Leung WT, Johnson PJ (2001) Systemic therapy for hepatocellular carcinoma. Semin Oncol 28:514–529 Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carcinoma. Lancet 362:1907–1917 Nagasue N, Kohno H, Chang YC, Taniura H, Yamanoi A, Uchida M et al (1993) Liver resection for hepatocellular carcinoma. Results of 229 consecutive patients during 11 years. Ann Surg 217:375–384 Nerenstone SR, Ihde DC, Friedman MA (1988) Clinical trials in primary hepatocellular carcinoma: current status and future directions. Cancer Treat Rev 15:1–31 Okano J, Nagahara T, Matsumoto K, Murawaki Y (2007) The growth inhibition of liver cancer cells by paclitaxel and the involvement of extracellular signal-regulated kinase and apoptosis. Oncol Rep 17:1195–1200 Palmer DH, Hussain SA, Johnson PJ (2004) Systemic therapies for hepatocellular carcinoma. Expert Opin Investig Drugs 13:1555–1568 Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108

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J Cancer Res Clin Oncol (2010) 136:267–274 Patt YZ, Charnsangavej C, YoVe B, Smith R, Lawrence D, Chuang V et al (1994) Hepatic arterial infusion of Xoxuridine, leucovorin, doxorubicin, and cisplatin for hepatocellular carcinoma: eVects of hepatitis B and C viral infection on drug toxicity and patient survival. J Clin Oncol 12:1204–1211 Pelletier G, Roche A, Ink O, Anciaux ML, Derhy S, Rougier P et al (1990) A randomized trial of hepatic arterial chemoembolization in patients with unresectable hepatocellular carcinoma. J Hepatol 11:181–184 Piccart-Gebhart MJ, Burzykowski T, Buyse M, Sledge G, Carmichael J, Lück HJ et al (2008) Taxanes alone or in combination with anthracyclines as Wrst-line therapy of patients with metastatic breast cancer. J Clin Oncol 26:1980–1986 Shi P, Wang MM, Jiang LY, Liu HT, Sun JZ (2008) Paclitaxel– doxorubicin sequence is more eVective in breast cancer cells with heat shock protein 27 overexpression. Chin Med J (Engl) 121:1975–1979 Strumberg D, Erhard J, Harstrick A, Klaassen U, Müller C, Eberhardt W et al (1998) Phase I study of a weekly 1 h infusion of paclitaxel in patients with unresectable hepatocellular carcinoma. Eur J Cancer 34:1290–1292 Tanaka H, Kubo S, Tsukamoto T, Shuto T, Takemura S, Yamamoto T et al (2005) Recurrence rate and transplantability after liver resection in patients with hepatocellular carcinoma who initially met transplantation criteria. Transplant Proc 37:1254–1256 Thorgeirsson SS, Grisham JW (2002) Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 31:339–346 World Health Organization (2008) Motality database. WHO statistical information system. Available at: http://www.who.int/whosis/. Accessed July 2008 Yamamoto S, Sato Y, Takeishi T, Hirano K, Kobayashi T, Watanabe T et al (2005) Successful surgical treatment for hepatocellular carcinoma and concomitant risky esophageal varices. Hepatogastroenterology 52:1083–1086 Yeo W, Mok TS, Zee B, Leung TW, Lai PB, Lau WY et al (2005) A randomized phase III study of doxorubicin versus cisplatin/ interferon alpha-2b/doxorubicin/Xuorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma. J Natl Cancer Inst 97:1532–1538 Zhang ZL, Liu ZS, Sun Q (2005) Anti-tumor eVect of thalidomide and paclitaxel on hepatocellular carcinoma in nude mice. Chin Med J (Engl) 118:1688–1694

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