Potential implications of GRP58 expression and susceptibility of cervical cancer to cisplatin and thymoquinone-based therapy

Figures & data

Table 1 Primers for amplification of GRP58, HPRT, and β-actin genes

Gene	Primer sequence	Accession number	Size (bp)
GRP58	5′-TGCTAAAGGAGAGAAGTTTG-3′ 5′-CTGCTACCACTACCTTTCA-3′	NM_005313.4	161
β-actin	5′-CATGTACGTTGCTATCCAGGC-3′ 5′-CTCCTTAATGTCACGCACGAT-3′	NM_001101.3	185
HPRT	5′-CGAGATGTGATGAAGGAGATG-3′ 5′-CCTGTTGACTGGTCATTACAA-3′	NM_000194.2	250

Abbreviation: bp, base pairs.

Table 2 Polymerase chain reaction protocol for GRP58, HPRT, and β-actin

Step	Temperature (°C)	Time	Number of cycles
Initial denaturation	95	5 minutes	1
Denaturation	95	20 seconds	40
Annealing*	58.3	30 seconds	40
Extension	70	5 seconds	40

Note:

* 60°C for annealing for GRP58 and β-actin, and 58.3°C for HPRT.

Figure 1 Dose-response curves for HeLa, SiHa, Vero, and 3T3 cells following treatment with cisplatin (i) and thymoquinone (ii) at 24 hours (A), 48 hours (B), and 72 hours (C). The cells (0.7×10⁵ mL⁻¹ for HeLa, 3T3, and Vero cells, and 1×10⁵ mL⁻¹ for SiHa cells) were treated with different concentrations of cisplatin and thymoquinone and subjected to MTT assay.

Notes: The experiment was carried out in triplicate. The results are shown as the mean ± standard deviation.
Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Table 3 Cytotoxicity of cisplatin and thymoquinone towards various human cell lines represented as IC₅₀ value determined by MTT assay

Cell line	Incubation time (hours)	IC50 (μM)
		Cisplatin	Thymoquinone
HeLa	24	38.55±1.81	119.24±1.90
	48	18.44±2.97*	72.1±3.81
	72	13.3±2.52*	29.57±5.81*
SiHa	24	37.78±7.46	87.76±0.34
	48	24.45±2.18*	52.31±4.96*
	72	19.5±2.12*	23.41±1.51*
3T3	24	39.77±2.79	70.64±0.19
	48	10.38±0.25*	69.3±13.75
	72	9.6±0.02*	61.67±5.01*
Vero	24	11.77±0.67	21.76±1.22
	48	9.72±0.02*	17.74±1.99
	72	9.65±0.08*	17.38±2.94*

Notes: The IC₅₀ is the average ± standard deviation value of three independent experiments.

* Significantly different from control at P<0.05.

Abbreviations: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC₅₀, half maximal inhibitory concentration.

Figure 2 Optimization of quantitative real-time polymerase chain reaction.

Notes: Single band with the correct product size (161 bp for GRP58, 250 bp for β-actin, and 185 bp for HPRT) at different temperatures as verified by 1% agarose gel electrophoresis (A). Representative amplification, standard and melting curves of primers for GRP58 (B–D). Single peak for each primer further validates the absence of primer dimers and nonspecific amplifications.
Abbreviations: bp, base pairs; RFU, relative fluorescence units.

Figure 3 mRNA level of GRP58 in HeLa and SiHa cells following treatment with cisplatin and thymoquinone for 48 hours as measured by quantitative real-time polymerase chain reaction.

Notes: (A) Cycle threshold values for HeLa and SiHa after treatment with cisplatin. The cycle threshold values for HeLa are lower, indicating a higher concentration of GRP58 as compared with SiHa. Treatment with cisplatin and thymoquinone downregulated GRP58 in HeLa cells (B) and SiHa cells (C). The data indicate relative levels of GRP58 that were normalized against β-actin and HPRT. Columns represent the mean ± standard deviation, each performed in triplicate. *P<0.05 was considered to be statistically significant.
Abbreviation: TQ, thymoquinone.

Figure 4 GRP58 expression in human cervical cancer cell lines treated with cisplatin and thymoquinone as determined by Western blot analysis.

Notes: Downregulation of GRP58 in HeLa cells (A) and SiHa cells (B) was observed following treatment with cisplatin and thymoquinone at two different time points. Relative levels of GRP58 were normalized against the loading control, β-actin.
Abbreviation: Con, control.

Figure 5 GRP58 levels in HeLa and SiHa cell lines following treatment with cisplatin (A) and thymoquinone (B).

Notes: GRP58 density was quantified by densitometric analysis. Data are shown as the mean ± standard deviation of at least three independent experiments. *P<0.05 versus negative (untreated) control.
Abbreviations: TQ, thymoquinone; h, hours.

Figure 6 Correlation between density values of the ratio of GRP58 to β-actin and cytotoxicity (IC₅₀ values) in HeLa and SiHa cells following treatment with cisplatin (A) and thymoquinone (B) using Pearson’s correlation method for at least three independent experiments. P<0.05 was considered to be statistically significant.

Abbreviations: TQ, thymoquinone; h, hours; IC₅₀, half maximal inhibitory concentration.

Figure 7 Schematic diagram of proposed mechanism of action for GRP58 in cisplatin-induced apoptosis. The pathways involve the endoplasmic reticulum stress-apoptotic-dependent pathway.

Abbreviations: NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; mTOR, mammalian target of rapamycin; PUMA, p53 upregulated modulator of apoptosis; JNK, C-Jun N-terminal kinase; Bcl-2, B-cell lymphoma 2; XBP1, X-box binding protein 1; CHOP, C/EBP-homologous protein; ATF, activating transcription factor 4; UPR, unfolded protein response; STAT, signal transducer and activator of transcription; GRP, glucose-regulated protein; TRAF2, TNF receptor-associated factor 2; IRE-1a, inositol-requiring protein 1.