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Cross-resistance to clinically used tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib.

Gotink KJ, Rovithi M, de Haas RR, Honeywell RJ, Dekker H, Poel D, Azijli K, Peters GJ, Broxterman HJ, Verheul HM - Cell Oncol (Dordr) (2015)

Bottom Line: The previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib.Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells.Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, VU University Medical Center, Rm 3A46, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT

Purpose: When during cancer treatment resistance to a tyrosine kinase inhibitor (TKI) occurs, switching to another TKI is often considered as a reasonable option. Previously, we reported that resistance to sunitinib may be caused by increased lysosomal sequestration, leading to increased intracellular lysosomal storage and, thereby, inactivity. Here, we studied the effect of several other TKIs on the development of (cross-) resistance.

Methods: TKI resistance was induced by continuous exposure of cancer cell lines to increasing TKI concentrations for 3-4 months. (Cross-) resistance was evaluated using MTT cell proliferation assays. Intracellular TKI concentrations were measured using LC-MS/MS. Western blotting was used to detect lysosome-associated membrane protein-1 and -2 (LAMP1/2) expression.

Results: The previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib. Exposure of 786-O and HT-29 cells to sorafenib, pazopanib or erlotinib for 3-4 months induced drug resistance to pazopanib and erlotinib, but not sorafenib. Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells. Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient. No cross-resistance to the mTOR inhibitor everolimus was detected.

Conclusions: Our data indicate that tumor cells can develop (cross-) resistance to TKIs, and that such resistance includes increased intracellular drug accumulation accompanied by increased lysosomal storage. Transient (cross-) resistance was found to occur for several of the TKIs tested, but not for everolimus, indicating that switching from a TKI to a mTOR inhibitor may be an attractive therapeutic option.

No MeSH data available.


Related in: MedlinePlus

Induction of resistance to sorafenib, pazopanib and erlotinib. (a) resistance patterns, determined by MTT proliferation assays, of the 786-O and HT-29 cells continuously exposed to sorafenib (SOR), pazopanib (PAZ) or erlotinib (ERL) for 3–4 months. (b) intracellular accumulation of sorafenib, pazopanib or erlotinib in parental (PAR) and continuously exposed SOR, PAZ and ERL cells. PAR cells were incubated for 24 h with drug-containing medium at the concentration of the continuous exposure. Drug concentrations: sorafenib: 3 μM; pazopanib: 20 μM; erlotinib: 20 μM. (c) Western blot analysis of lysosome-associated membrane protein-1 and −2 (LAMP-1 and −2), as a measure of the lysosomal compartment. (d) quantification of LAMP-1 and −2 by Western blot analysis. LAMP-1 and LAMP-2 expression was corrected for β-actin expression, and normalized to untreated samples (PAR). P values are derived from comparison to the PAR cell line. Results are shown as mean ± SEM. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001
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Fig3: Induction of resistance to sorafenib, pazopanib and erlotinib. (a) resistance patterns, determined by MTT proliferation assays, of the 786-O and HT-29 cells continuously exposed to sorafenib (SOR), pazopanib (PAZ) or erlotinib (ERL) for 3–4 months. (b) intracellular accumulation of sorafenib, pazopanib or erlotinib in parental (PAR) and continuously exposed SOR, PAZ and ERL cells. PAR cells were incubated for 24 h with drug-containing medium at the concentration of the continuous exposure. Drug concentrations: sorafenib: 3 μM; pazopanib: 20 μM; erlotinib: 20 μM. (c) Western blot analysis of lysosome-associated membrane protein-1 and −2 (LAMP-1 and −2), as a measure of the lysosomal compartment. (d) quantification of LAMP-1 and −2 by Western blot analysis. LAMP-1 and LAMP-2 expression was corrected for β-actin expression, and normalized to untreated samples (PAR). P values are derived from comparison to the PAR cell line. Results are shown as mean ± SEM. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001

Mentions: In order to investigate whether the resistant phenotype induced by sunitinib may represent an universal resistance mechanism, we exposed 786-O PAR and HT-29 PAR cells continuously for 3–4 months to increasing concentrations of the multi-targeted TKIs sorafenib and pazopanib and to the single-targeted TKI erlotinib to compare resistance induction to an EGFR family TKI. The induction of resistance by these three TKIs was found to be comparable to their cross-resistance phenotype in the sunitinib-selected cells: a prominent resistance to pazopanib and erlotinib (RF ≥ 3.1 - > 25) and no development of resistance to sorafenib (RF = 1.2; Fig. 3a and Table 2). The final concentration after 3–4 months of exposure was 3 μM sorafenib and 20 μM pazopanib or erlotinib, respectively. Higher concentrations of pazopanib and erlotinib could not be achieved due to their limited solubility in culture medium. Pazopanib- or erlotinib-resistant cells, as well as sorafenib-exposed cells, did not show cross-resistance to any of the other drugs tested (i.e., sunitinib, sorafenib, pazopanib, erlotinib, lapatinib, everolimus; data not shown). The intracellular accumulation of the selected TKIs (sorafenib, pazopanib, erlotinib) was measured in these continuously exposed cells and compared to shortly exposed parental cells. By doing so, a ~ 5–50 fold increase in pazopanib and erlotinib content, but not in sorafenib content, was found in the continuously exposed cells compared to the parental cells (Fig. 3b).Fig. 3


Cross-resistance to clinically used tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib.

Gotink KJ, Rovithi M, de Haas RR, Honeywell RJ, Dekker H, Poel D, Azijli K, Peters GJ, Broxterman HJ, Verheul HM - Cell Oncol (Dordr) (2015)

Induction of resistance to sorafenib, pazopanib and erlotinib. (a) resistance patterns, determined by MTT proliferation assays, of the 786-O and HT-29 cells continuously exposed to sorafenib (SOR), pazopanib (PAZ) or erlotinib (ERL) for 3–4 months. (b) intracellular accumulation of sorafenib, pazopanib or erlotinib in parental (PAR) and continuously exposed SOR, PAZ and ERL cells. PAR cells were incubated for 24 h with drug-containing medium at the concentration of the continuous exposure. Drug concentrations: sorafenib: 3 μM; pazopanib: 20 μM; erlotinib: 20 μM. (c) Western blot analysis of lysosome-associated membrane protein-1 and −2 (LAMP-1 and −2), as a measure of the lysosomal compartment. (d) quantification of LAMP-1 and −2 by Western blot analysis. LAMP-1 and LAMP-2 expression was corrected for β-actin expression, and normalized to untreated samples (PAR). P values are derived from comparison to the PAR cell line. Results are shown as mean ± SEM. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001
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Related In: Results  -  Collection

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Fig3: Induction of resistance to sorafenib, pazopanib and erlotinib. (a) resistance patterns, determined by MTT proliferation assays, of the 786-O and HT-29 cells continuously exposed to sorafenib (SOR), pazopanib (PAZ) or erlotinib (ERL) for 3–4 months. (b) intracellular accumulation of sorafenib, pazopanib or erlotinib in parental (PAR) and continuously exposed SOR, PAZ and ERL cells. PAR cells were incubated for 24 h with drug-containing medium at the concentration of the continuous exposure. Drug concentrations: sorafenib: 3 μM; pazopanib: 20 μM; erlotinib: 20 μM. (c) Western blot analysis of lysosome-associated membrane protein-1 and −2 (LAMP-1 and −2), as a measure of the lysosomal compartment. (d) quantification of LAMP-1 and −2 by Western blot analysis. LAMP-1 and LAMP-2 expression was corrected for β-actin expression, and normalized to untreated samples (PAR). P values are derived from comparison to the PAR cell line. Results are shown as mean ± SEM. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001
Mentions: In order to investigate whether the resistant phenotype induced by sunitinib may represent an universal resistance mechanism, we exposed 786-O PAR and HT-29 PAR cells continuously for 3–4 months to increasing concentrations of the multi-targeted TKIs sorafenib and pazopanib and to the single-targeted TKI erlotinib to compare resistance induction to an EGFR family TKI. The induction of resistance by these three TKIs was found to be comparable to their cross-resistance phenotype in the sunitinib-selected cells: a prominent resistance to pazopanib and erlotinib (RF ≥ 3.1 - > 25) and no development of resistance to sorafenib (RF = 1.2; Fig. 3a and Table 2). The final concentration after 3–4 months of exposure was 3 μM sorafenib and 20 μM pazopanib or erlotinib, respectively. Higher concentrations of pazopanib and erlotinib could not be achieved due to their limited solubility in culture medium. Pazopanib- or erlotinib-resistant cells, as well as sorafenib-exposed cells, did not show cross-resistance to any of the other drugs tested (i.e., sunitinib, sorafenib, pazopanib, erlotinib, lapatinib, everolimus; data not shown). The intracellular accumulation of the selected TKIs (sorafenib, pazopanib, erlotinib) was measured in these continuously exposed cells and compared to shortly exposed parental cells. By doing so, a ~ 5–50 fold increase in pazopanib and erlotinib content, but not in sorafenib content, was found in the continuously exposed cells compared to the parental cells (Fig. 3b).Fig. 3

Bottom Line: The previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib.Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells.Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, VU University Medical Center, Rm 3A46, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT

Purpose: When during cancer treatment resistance to a tyrosine kinase inhibitor (TKI) occurs, switching to another TKI is often considered as a reasonable option. Previously, we reported that resistance to sunitinib may be caused by increased lysosomal sequestration, leading to increased intracellular lysosomal storage and, thereby, inactivity. Here, we studied the effect of several other TKIs on the development of (cross-) resistance.

Methods: TKI resistance was induced by continuous exposure of cancer cell lines to increasing TKI concentrations for 3-4 months. (Cross-) resistance was evaluated using MTT cell proliferation assays. Intracellular TKI concentrations were measured using LC-MS/MS. Western blotting was used to detect lysosome-associated membrane protein-1 and -2 (LAMP1/2) expression.

Results: The previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib. Exposure of 786-O and HT-29 cells to sorafenib, pazopanib or erlotinib for 3-4 months induced drug resistance to pazopanib and erlotinib, but not sorafenib. Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells. Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient. No cross-resistance to the mTOR inhibitor everolimus was detected.

Conclusions: Our data indicate that tumor cells can develop (cross-) resistance to TKIs, and that such resistance includes increased intracellular drug accumulation accompanied by increased lysosomal storage. Transient (cross-) resistance was found to occur for several of the TKIs tested, but not for everolimus, indicating that switching from a TKI to a mTOR inhibitor may be an attractive therapeutic option.

No MeSH data available.


Related in: MedlinePlus