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- Materials and Methods
To examine the mechanism of resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in lung cancer, we continuously exposed the non-small-cell lung cancer (NSCLC) cell line NCI-H23 to SN-38 and selected the SN-38-resistant clone H23/SN-38. After 2 months of culturing in SN-38-free conditions, H23/SN-38 cells recovered their sensitivity to SN-38 and were subsequently established as the revertant H23/SN-38REV cell line. Because H23/SN-38 cells show cross resistance to certain anticancer drugs, such as topotecan, etoposide, doxorubicin and mitoxantrone, we examined the gene and protein expression levels of drug efflux transporters of the ATP-binding cassette (ABC) family. We found that both gene and protein expression of ABCG2/BCRP (ABCG2) in H23/SN-38 cells was increased compared with that in NCI-H23 cells and H23/SN-38REV cells. The cellular accumulation of topotecan in H23/SN-38 cells was decreased compared with that in NCI-H23 and H23/SN-38REV cells, and treatment with reserpine (an inhibitor of ABCG2) increased the cellular accumulation of topotecan in H23/SN-38 cells. Furthermore, treatment with reserpine also altered the sensitivity of H23/SN-38 cells to SN-38. These results indicate that the upregulation of ABCG2 was functional, and related to the resistance of H23/SN-38 cells to SN-38. Moreover, we found that gene expression levels of ABCG2 were significantly correlated with the concentration of SN-38 for 50% cell survival in 13 NSCLC cells (r = 0.592, P < 0.05). The present results indicate that the induction of ABCG2 by SN-38 does confer acquired resistance to CPT-11/SN-38, but the induction of ABCG2 and subsequent drug resistance are reversible. However, the expression level of ABCG2 may be a useful indicator of CPT-11/SN-38 activity in lung cancer. (Cancer Sci 2006; 97: 192–198)
7-Ethyl-10-hydroxycamptothecin (SN-38) is the active metabolite of irinotecan hydrochloride (CPT-11), one of the topoisomerase inhibitors. Several mechanisms of resistance to CPT-11/SN-38 have been reported. A point mutation of the DNA topoisomerase I gene has been described in vitro(1) but no alteration of DNA topoisomerase I gene was observed in vivo, and low DNA topoisomerase I expression is associated with resistance to CPT-11/SN-38.(2) SN-38 has been shown to undergo glucuronidation by uridine 5′diphosphoglucuronosyltransferase (UGT) to form inactive SN-38-glucuronide, and hence increased intracellular drug detoxification through the upregulation of glucuronidation activity catalyzed by UGT is reportedly associated with resistance to CPT-11/SN-38.(3–5) Furthermore, it has also been suggested that reduced drug accumulation mediated by the ATP-dependent efflux pump is involved in the mechanism of resistance to CPT-11/SN-38.(6–8)
Lung cancer is one of the most common malignancies worldwide, and several randomized clinical trials and meta-analyses have demonstrated that chemotherapy can slightly but significantly prolong survival in patients with lung cancer.(9,10) CPT-11 is one of the promising agents against human lung cancers(11,12) and resistance to CPT-11 is a common problem in the survival of lung cancer patients. However, biomarkers for predicting resistance to CPT-11 have not been fully identified. To elucidate the biomarkers of resistance to CPT-11, we developed an in vitro model of resistance to SN-38 by continuous and progressive exposure of the non-small-cell lung cancer (NSCLC) cell line NCI-H23 to SN-38, and selected the SN-38-resistant clone H23/SN-38. We then cultured H23/SN-38 cells under SN-38-free conditions for 2 months and subsequently established the revertant H23/SN-38REV cell line that exhibited sensitivity to SN-38. Here we describe the determinants of resistance to CPT-11/SN-38 as elucidated using our selected SN-38-resistant cells.
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- Materials and Methods
In the present study we successfully isolated an SN-38-resistant clone (H23/SN-38) by continuously exposing NCI-H23 cells to SN-38. The H23/SN-38 cells showed enhancement of functional ABCG2 expression, which demonstrates that overexpression of ABCG2 is related to the acquired resistance to SN-38 in lung cancer.
Multidrug resistance is often attributed to ABC transporters in cancer cells. ABCB1 was the first of the ABC transporters to be identified and characterized, and the isolation of the second distantly related ABCC1 transporter facilitated the discovery of eight more genes. ABCG2 belongs to another ABC transporter family, and has its own characteristic substrate specificity and biology. ABCB1 confers high levels of resistance to bulky amphipathic drugs. The spectrum of drugs affected by ABCC transporters is similar to that for ABCB1; however, ABCC transporters efflux anticancer drugs by cotransporting drugs with glutathione, or glutathione–drug or glucuronide–drug conjugates. In contrast, ABCG2 affects a narrower range of anticancer agents compared to the ABCB1 and ABCC transporters, including anthracyclines, mitoxantrone and topoisomerase I inhibitors.(17) Our finding that H23/SN-38 cells that overexpress ABCG2 showed cross-resistance to topotecan, mitoxantrone, doxorubicin and etoposide − but not to paclitaxel, vinorelbine, 5-fluorouracil or cisplatin − is similar to the ABCG2 drug-resistance profiles in previous reports.
It has been reported recently that overexpression of ABCG2 directly confers resistance to SN-38 in lung cancer cells.(8) SN-38 undergoes glucuronidation by UGT to form inactive SN-38-glucuronide, and ABCC2 effluxes mainly SN-38-glucuronide products and those related with CPT-11/SN-38 resistance.(6,18) In contrast, ABCG2 transports both SN-38 and SN-38-glucuronide products with a higher affinity for SN-38.(19) We did not detect ABCC2 gene expression in H23/SN-38 cells. Furthermore, we did not detect gene expression of UGT1A − which is mainly involved in SN-38 glucuronidation − in H23/SN-38 cells. These results indicate that overexpression of ABCG2 may be sufficient to confer resistance to SN-38 through efflux of SN-38 itself.
The mechanism underlying the upregulation of ABCG2 is still uncertain. ABCC family transporters confer resistance to methotrexate for only short-term exposure to this drug. In contrast, ABCG2 transports the polyglutamylated forms of methotrexate and confers resistance to prolonged exposure to this agent.(17,20) Candeil et al.(21) also hypothesize − from their in vitro and in vivo experiments − that in colon cancer a longer exposure to CPT-11 can lead to a larger proportion of ABCG2-overexpressing cells in the tissue samples. These results indicate that short-term exposure to anticancer drugs does not affect ABCG2 expression and that the mechanism underlying the upregulation of ABCG2 may be quite different from those of other ABC transporters.
After 2 months culturing under SN-38-free conditions, H23/SN-38 cells recovered their sensitivity to SN-38 concomitant with a decreased expression of ABCG2. To determine whether this phenomenon was also present in other established SN-38-resistant cells, we cultured the SN-38-resistant SCLC cell line PC-6/SN2-5 under SN-38-free conditions for 2 months, which resulted in ABCG2 expression decreasing concomitant with a decreased resistance to SN-38. We believe this to be the first report that the induction of ABCG2 by anticancer drugs is reversible; the present results indicate that the effect of ABCG2 on the acquired resistance to CPT-11/SN-38 is reversible. However, we found that resistance to SN-38 does not recover completely when ABCG2 expression decreased. A similar result was obtained following treatment with an ABCG2 inhibitor, which failed to restore the complete sensitivity of SN-38-resistant colon cancer cells to SN-38.(21) Taken together, the above results indicate that the mechanism of acquired resistance to SN-38 is multifactorial. Indeed, CPT-11 activates transcriptional factor nuclear factor (NF)-κB, which plays an important role for underlying mechanisms of the antiapoptotic pathway and inducible drug resistance.(22) Therefore, further studies are required to elucidate the determinants in relation to the persistent acquired resistance to SN-38. Nonetheless, the recovery of ABCG2 expression may be an important concept for determining lung cancer chemotherapies, because we are able to use ABCG2-mediated drugs when patients are pretreated with CPT-11-containing chemotherapy at least a few months prior.
We also found a correlation between the IC50 for SN-38 and ABCG2 expression in 13 NSCLC cells. There is reportedly a strong correlation between mRNA and protein expression levels for ABCG2 in breast cancer.(23) Moreover, the gene expression levels of ABCG2 reportedly reflect the functional activity of ABCG2 in lung cancer.(15) These results indicate that the expression levels of ABCG2 may be a useful indicator of CPT-11/SN-38 activity in lung cancer.
Many substrates and inhibitors against ABCG2 have been reported.(24) In the present study, we found that the ABCG2 inhibitor reserpine modified the resistance of H23/SN-38 cells to SN-38. The epidermal growth factor receptor kinase inhibitor gefitinib and some flavonoids have recently been shown to act as ABCG2-substrate anticancer drugs, and coadministration of these agents with ABCG2-inhibitors increases the toxicity for cancers.(25,26) Because ABCG2 is expressed in many types of cancers(27) treatment with ABCG2-inhibitors may be useful to overcome ABCG2-mediated drug resistance in cancer treatments. Whereas ABCG2 expression in normal tissues is also found in placental syncytiotrophoblasts, the epithelia of the small intestine and colon, the liver canalicular membrane, and in ducts and lobules of the breast,(27) the physiological role of ABCG2 in normal tissues remains uncertain. It is therefore necessary to consider possible toxicity when substrates or inhibitors to ABCG2 are used in cancer chemotherapy.
In conclusion, the present study shows that the induction of ABCG2 by SN-38 does not confer permanent acquired resistance to CPT-11/SN-38. However, the expression level of ABCG2 may be a useful determinant for CPT-11/SN-38 activity in lung cancer. Future studies should investigate the clinical and functional involvement of ABCG2 in resistance to CPT-11/SN-38 in vivo.