Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells

Abstract

There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.

Protein profiling of WERI RB1 and etoposide resistant WERI ETOR reveals new insights into topoisomerase inhibitor resistance in retinoblastoma

“…The etoposide sensitive and resistant RB cell lines WERI RB1 and WERI ETOR were cultivated as previously described (13)(14)(15)(16)(17). The identities of these RB cell lines were verified by DNA fingerprinting and profiling using eight different and highly polymorphic short tandem repeat (STR) loci (Leibniz-Institute DSMZ GmbH, Braunschweig, Germany).…”

“…Furthermore, Busch et al demonstrated a higher proliferation rate in WERI ETOR as well as an increased tumor formation and tumor size in an in vivo chick chorioallantoic membrane assay (17). Mergler et al and Oronowicz et al showed alterations in thermosensitive transient receptor potential channels in WERI ETOR cells (13,16). A publication by Kakkassery et al proved an upregulation of sphingosine-1-phosphate in the WERI ETOR cell line as a potential resistance mechanism in RB (14).…”

“…To identify the mechanisms of chemotherapy resistance, the etoposide resistant subclone WERI ETOR was established by harvesting surviving cells after incubation with increasing etoposide doses from WERI RB1 as described by Stephan et al (12). Previous studies also examined the parental WERI RB1 and the etoposide resistant subclone WERI ETOR on the molecular level (13)(14)(15)(16)(17)(18). Furthermore, Busch et al demonstrated a higher proliferation rate in WERI ETOR as well as an increased tumor formation and tumor size in an in vivo chick chorioallantoic membrane assay (17).…”

Protein Profiling of WERI-RB1 and Etoposide-Resistant WERI-ETOR Reveals New Insights into Topoisomerase Inhibitor Resistance in Retinoblastoma

“…To identify the mechanisms of chemotherapy resistance, the etoposide-resistant subclone WERI-ETOR was established by harvesting surviving cells after incubation with increasing etoposide doses from WERI-RB1, as described by Stephan et al [ 12 ]. Previous studies also examined the parental WERI-RB1 and the etoposide-resistant subclone WERI-ETOR on the molecular level [ 13 , 14 , 15 , 16 , 17 , 18 ]. Furthermore, Busch et al demonstrated a higher proliferation rate in WERI-ETOR as well as increased tumor formation and tumor size in an in vivo chick chorioallantoic-membrane assay [ 17 ].…”

Calcium Regulation on the Atrial Regional Difference of Collagen Production Activity in Atrial Fibrogenesis

“…Soluble guanylyl cyclase, a messenger that can decrease collagen production and Ca 2+ entry, is highly expressed in RA tissues but markedly reduced or absent in LA tissues [ 18 , 19 , 20 ]. Compared with RA fibroblasts, LA fibroblasts produce a higher level of oxidative stress, which has been found to induce Ca 2+ entry [ 13 , 21 ]. Increased intracellular Ca 2+ is mainly derived either from extracellular Ca 2+ entry or from endoplasmic reticulum (ER) Ca 2+ release.…”