Treatment of resistant human colon cancer xenografts by a fluoxetine–doxorubicin combination enhances therapeutic responses comparable to an aggressive bevacizumab regimen
Introduction
Surgery followed by drug therapy is standard treatment for colorectal cancer – the third most common form of cancer and the second leading cause of cancer-related death in the Western world [1], [2]. Recently, a novel modality utilizing drugs that target tumor vasculature, such as bevacizumab (avastin), was added to the veteran modality of conventional chemotherapeutic drugs [3], [4], [5], [6], [7], [8], [9], [10]. Frequently-administered combinations include: fluorouracil, doxorubicin and mitomycin C or methotrexate; etoposide, doxorubicin and cisplatin; docetaxel, cisplatin with/without fluorouracil [6], [7], [8], [9], [10]. The veteran modality is prone, however, to multiple drug resistance (MDR) operated by the ABC transporters ABCB1 (P-glycoprotein, Pgp), members of the ABCC family (multidrug resistance-associated proteins, MRP) and ABCG2 (breast cancer resistance protein, BCRP) [11], [12], [13], [14], [15], [16], [17], [18], [19]. Doxorubicin, mitomycin C and docetaxel are known substrates of Pgp and doxorubicin is also a substrate of MRP1 [12], [13], [15], [16], [17].
Drug resistance operated by the ABC transporters is an influx–efflux imbalance. The MDR transporters actively pump their substrates out of the cell, reducing intracellular drug doses below lethal thresholds [11], [13], [15]. A major approach to correct this imbalance is by pump inhibition, utilizing chemosensitizers that would be administered together with the anti-cancer drugs [11], [13], [15], [17], [20], [21]. Currently, several third-generation chemosensitizers, mostly against Pgp, are in clinical trials [17], [22]. However, few trials are currently conducted, and the debate on this strategy is still going on. In the chemosensitizer arena, most pre-clinical studies have focused, in vitro and in vivo, on systems exhibiting high levels of drug resistance which is technically advantageous [23], [24]. For patients, moderate resistance is already a severe therapeutic impediment, that is often further aggravated after exposure to chemotherapy [25], [26], [27]. A recommended strategic approach is to apply a combination of chemotherapy and chemosensitizer at the beginning of cancer treatment when resistance is still low or moderate [27]. To gain more insight into modulating clinically-relevant resistance levels, there is an obvious need to also examine chemosensitization in pre-clinical moderate-resistance systems. Such examination should include functional and mechanistic aspects, assessing whether the level of resistance affects patterns and quantitative measures of resistance modulation.
In this manuscript, we focused on pre-clinical studies in the HCT-15 cell line derived from human colorectal adenocarcinoma, a system postulated to model moderate resistance in colorectal cancer. HCT-15 is reported to be an inherent MDR line expressing moderate levels of Pgp [28], [29], [30]. In one study, it was also reported to moderately-express MRP [31]. Consequently, we first affirmed that the cells we studied were a Pgp-alone line.
The purpose of the in vitro functional and mechanistic studies was 2-fold: (i) To characterize MDR in this cell line selecting doxorubicin, a frequent component of chemotherapy combinations for colorectal cancer and a Pgp substrate, as the test drug [6], [8], [9], [10], [15], [16], [17], [20]. (ii) To modulate this resistance by chemosensitization, investigating fluoxetine (Prozac), previously shown by us to act as a chemosensitizer for highly-resistant Pgp-expressing cancer cells, as the test chemosensitizer [20], [21]. The purpose of the in vivo studies conducted in an established animal model of HCT-15 xenografts [31], [32], [33], was also 2-fold: (i) To determine whether the functional results obtain in vitro come into expression in vivo, which is (obviously) a critical pre-clinical step. (ii) To compare the two treatment modalities discussed above for colorectal cancer, i.e. chemotherapy and bevacizumab, under conditions where resistance to chemotherapy may be significantly reduced. As will be shown, modulating doxorubicin resistance by fluoxetine makes this combination a modern-day therapeutic option worthy of consideration for colon cancer.
Section snippets
Materials
Doxorubicin was from Teva Pharmaceutical Inc. (Netanya, Israel), bevacizumab and fluoxetine were a kind gift from Teva Pharmaceutical Inc. (Netanya, Israel). Bovine serum albumin (BSA), sodium azide, sodium chloride and hepes were from Sigma (St. Louis, USA). Materials for cell culture, phosphate buffered saline (PBS) and XTT kit were from Biological industries (Beit Haemek, Israel).
Expression of ABC transporters
Although HCT-15 cells are known to be an inherent-Pgp cell line [28], [29], [30], [31], due to the dynamic nature of cells in culture, we found it imperative to affirm prior to any MDR-related studies, that the specific cell batches we studied expressed Pgp. Since in one case, presence of MRP in these cells was reported as well, we also tested for the other two major transporter types – MRP and BCRP – known to be involved in MDR. Typical results of flow cytometry studies, using antibodies
Discussion
For cancer patients MDR is a major impediment, whether the resistance is moderate or severe. Pre-clinical investigations into the resistance and its modulation are frequently performed utilizing cells that express very high levels of resistance. In the present study we deliberately focused on moderate resistance. The selected HCT-15 line, originating from human colorectal cancer, is classified as expressing Pgp [28], [29], [30] and in one reported to also express MRP [31]. We tested for all the
Conflicts of interest statement
None declared.
Acknowledgements
We thank Orit Sagi-Assif for her insights into, and assistance with, the flow cytometry studies and Alexander Barbul for his valuable contributions to data acquisition and analysis in confocal microscopy.
References (43)
- et al.
Colorectal cancer screening in elderly patients: when should be more useful?
Cancer Treat. Rev.
(2007) - et al.
Adjuvant chemotherapy with 5-fluorouracil, doxorubicin and mitomycin-C (FAM) for 6 months after curative resection of gastric carcinoma
Eur. J. Surg. Oncol.
(2007) - et al.
Inhibition of P-glycoprotein (ABCB1)- and multidrug resistance-associated protein 1 (ABCC1)-mediated transport by the orally administered inhibitor, CBT-1®
Biochem. Pharmacol.
(2008) - et al.
Multidrug resistance mediated by the multidrug resistance protein (MRP) gene
Biochem. Pharmacol.
(1996) - et al.
Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense
Toxicol. Appl. Pharmacol.
(2005) - et al.
Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions?
Biochem. Pharmacol.
(2004) - et al.
Gomisin A alters substrate interaction and reverses P-glycoprotein-mediated multidrug resistance in HepG2-DR cells
Biochem. Pharmacol.
(2006) - et al.
Fluoxetine and reversal of multidrug resistance
Cancer Lett.
(2006) - et al.
Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells
Biochem. Biophys. Res. Commun.
(2007) - et al.
Incidence of P-glycoprotein overexpression and multidrug resistance (MDR) reversal in adult soft tissue sarcoma
Eur. J. Cancer
(2000)
Differential anthracycline sensitivity in two related human colon carcinoma cell lines expressing similar levels of P-glycoprotein
Cancer Lett.
Reversal of multidrug resistance-associated protein-mediated daunorubicin resistance by camptothecin
J. Pharm. Sci.
Synthesis and in vitro evaluation of 1,8-diazaanthraquinones bearing 3-dialkylaminomethyl or 3-(N-alkyl- or N-aryl)carbamoyloxymethyl substituent
Eur. J. Med. Chem.
Surgical treatment for digestive cancer. Current issues – colon cancer
Dig. Surg.
Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer
N. Engl. J. Med.
Integration of novel agents in the treatment of colorectal cancer
Cancer Chemother. Pharmacol.
Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro
Angiogenesis
Docetaxel, cisplatin, and fluorouracil; docetaxel and cisplatin; and epirubicin, cisplatin, and fluorouracil as systemic treatment for advanced gastric carcinoma: a randomized phase II trial of the Swiss Group for Clinical Cancer Research
J. Clin. Oncol.
Neoadjuvant therapy for patients with locally advanced gastric carcinoma with etoposide, doxorubicin, and cisplatinum. Closing results after 5 years of follow-up
Cancer
Randomized trial comparing epirubicin, cisplatin, and fluorouracil versus fluorouracil, doxorubicin, and methotrexate in advanced esophagogastric cancer
J. Clin. Oncol.
Sequential high-dose methotrexate and fluorouracil combined with doxorubicin – a step ahead in the treatment of advanced gastric cancer: a trial of the European Organization for Research and Treatment of Cancer Gastrointestinal Tract Cooperative Group
J. Clin. Oncol.
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Present address: Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA.