Since cancer is the second leading cause of death worldwide, research is ongoing to develop effective therapeutic strategies with few side effects. Furthermore, many chemotherapeutics such as cisplatin have shown decreased effectiveness due to epigenetic dynamics and genetic changes, leading to the mechanism of multidrug resistance (MDR). The ATP-Binding Cassette (ABC) transporter participates in MDR as overexpression of this protein potentially increases drug molecule efflux, preventing many anticancer drugs from reaching their intracellular targets. All ABC transporters are expressed in normal cells, such as ABCC1 which is highly expressed in normal lung cells. ABC transporters play a role in normal metabolic processes as well as protection against environmental cues and stress signals (MDR1 inducers). Some of the transporters confer drug resistance to human tumors, expressing in a tissue-restricted manner, play important role in metabolic processes and cellular signaling, while the expression in the tissue of origin exhibits lower expression. In addition, alterations in the expression of growth proteins, oncogenes, and death-controlling proteins have been reported to influence the transcription and expression of drug-resistant genes, such as MDR1 or Multidrug Resistant Protein (MRP).
Lung cancer is one of the most common causes of mortality worldwide due to the development of resistance to chemotherapeutics such as cisplatin. It has been reported that the overexpression of several ABC transporter coding genes, such as ABCB1, ABCC1, ABCC2, ABCC3, and ABCG2,[9–12] leads to decreased sensitivity to cisplatin or cis-diamine-dichloroplatinum (II), a platinum-based drug used to treat many cancers. Cisplatin acts by binding to the purine base of the DNA, inhibiting transcription and translation, and finally leading to apoptosis. Theoretically, the activity of ABC transporters can cause cisplatin to be removed from cells before it can bind to DNA thus higher doses are required to achieve effective inhibition. However, high doses of cytotoxic-based drugs, such as cisplatin, often cause side effects in normal cells. A combination treatment strategy employing Cisplatin with other compounds, such as those natural products, can potentially reduce the dose of chemotherapy drugs, reducing toxicity and improving the therapeutic effect.
Among natural products that has been well reviewed to share some promises to improve therapeutic effect of cisplatin includes class of flavonoids, saponins, alkaloids, polysaccharides, phenylpropanoids, and napthoquinones. Bioactive carotenoids remain understudied yet possessing a unique chemical structure and exhibit broad pharmacological effects, including anticancer activity. The carotenoid fucoxanthin has been reported to be potential ABC transporter substrate that demonstrated more effective inhibitory activity against P-glycoprotein than verapamil in doxorubicin-resistant human T cell lymphoma (CEM/ADR5000) and colon cancer cells (Caco-2).[16,17] Furthermore, carotenoids may be effective against various types of cancer cells due to the diversity of their structure affecting their biological activity. In addition, the genetic diversity of cancer cells provides type-specificity consequences, thus affecting their susceptibility to cancer therapy.[19,20] Previously, we demonstrated the combination of bixin or fucoxanthin with cisplatin [Figure 1] exerted synergistic effects in A549 lung cancer cells compared to HeLa cervical cancer cells which express fewer ABC transporters than lung cancer cells.
The expression of ABC transporters is highly regulated, especially at the transcription level, possibly because of the varied physiological functions of these different transporters. The regulation requires the complex interplay of factors involved in differentiation and development, as well as those associated with cell survival and apoptosis elicited upon intrinsic and environmental stress.[6,20] At the protein level, several MRPs are involved in the transport of negatively charged anionic drugs and neutral drugs conjugated to glutathione, glucuronate, or sulfate.[21,22] Activation of MDR 1 was also associated with cell damage, suggesting that increased Pgp transcription may be involved in the cellular response to damaging agents.[23,24] The overexpression of MDR1 in response to chemotherapeutics is due to changes in mRNA stabilization and translational initiation in several leukemia cell lines, suggesting that multiple mechanisms exist in different cell types, either cooperatively or exclusively regulate the transcription of MDR genes.
The wild-type tumor suppressor protein p53 can repress transcription of the MDR1 gene by binding to a novel binding element within the proximal MDR1 promoter (−72 to −40) which induces a tetrameric conformation to transform p53 from an activator to a repressor. It has been suggested that activation of p53 on the murine mdr1 promoter occurs in response to DNA damage and stress, whereas down-regulated p53 expression is associated with increased MRP1 expression in colorectal cancer.[28,29] The p53 regulation of drug-resistance genes is complex and not well understood.[30,31] Thus, more research is required to investigate variations in the individual promoters, endogenous p53, the presence or absence of other p53 family members, and cell- and tissue-specific co-effectors of p53 activity.
The current study investigated the regulation of ABCC1 and ABCC2, as well as the tumor suppressor corresponding gene, p53, in response to carotenoids alone and in combination with cisplatin in A549 lung cancer cell lines.
MATERIALS AND METHODS
Chemical and cell lines
The A549 human lung cancer cell line was obtained from American Type Culture Collection (ATCC). Culture media, supplements, and antibiotics were purchased from PAN-Biotech (Aidenbach, Germany) and trypsin was from Sisco Research Laboratories Pvt. Ltd. (SRL) (Mumbai, India). Cisplatin, Bixin, and Fucoxanthin were purchased from Sigma-Aldrich (Missouri, United States). The Total RNA Mini Kit (Blood/Cultured Cell) was purchased from Geneaid Biotech Ltd. (New Taipei City, Taiwan Republic of China) and the SensiFAST™ SYBR® No-ROX One-Step Kit was from Meridian Bioscience (Ohio, United States). The primers ABCC1 (F 5′-ATGTCACGTGGAATACCAGC-3′ and R 5′-GAAGACTGAACTCCCTTCCT-3′), ABCC2 (F 5′-AGTGAATGACATCTTCACGTTTG-3′ and R 5′-CTTGCAAAGGAGATCAGCAA-3′), p53 (F 5′-CCCTCCTCAGCATCTTATCCG-3′ and R 5′-CAACCTCAGGCGGCTCATAG-3′), and caspase 3 (F 5′-TTAATAAAGGTATCCATGGAGAACACT-3′ and R 5′-TTAGTGATAAAAATAGAGTTCTTTTGTGAG-3′) were purchased from PT. Genetika Science (Indonesia, Banten, Indonesia). All laboratory plastic ware was obtained from Wuxi Nest Biotechnology Co., Ltd (Jiangsu, China).
A549 cells were grown in 10% DMEM with Glutamax and antibiotics (500 U/mL penicillin and 500 g/mL streptomycin) and maintained in culture media without NEAA. All cells were grown at 37°C and 5% CO2.
A549 cells was treated with each compound alone (cisplatin, fucoxanthin, and bixin) and with combination between cisplatin and carotenoids, either fucoxanthin or bixin using a constant ratio with ED50 from each compound, based on our previous study [Table 1]. The method was developed according to previous studies.[32,33] In brief, 3 × 105 cells were grown in each well of 6-well plates for 24 h. Compound alone or compounds in combination was then added in the designed cells, following with the incubation for 48h at 37°C with continuous 5% CO2. RNA from the treated and untreated cells were then isolated.
Real-time quantitative-polymerase chain reaction (RT-qPCR)
Total RNA was isolated from treated and untreated A549 lung cancer cells using the Total RNA Mini Kit according to the manufacturer’s instructions. The concentration was determined using the Nanodrop (Thermo Fischer Scientific) at 260 nm and 280 nm. RT-qPCR was performed using 100 picogram/mL of RNA and the SensiFAST™ SYBR® No-ROX One-Step Kit according to the manufacturer’s protocol. Briefly, the thermal cycler machine was set at 45°C for 10 minutes for reverse transcription to be carried out in the first cycle. After that, the reaction was carried out at 95rpm for 2 min for polymerase enzyme activation, then 95°C for 15 s for denaturation, 58°C–60°C for 30 s for annealing, and 72°C for 30 s for elongation. All the steps were carried out for 40 repeated cycles. The relative gene expression of ABCC1, ABCC2, caspase-3, and p53 was using the ∆Ct method by normalizing the cells without treatment so that the relative expression value is obtained.
ΔCt = Ct of gene interest - Ct of gene reference
Relative Expression = 2ΔCt
The Ct values were analyzed using a relative quantitative approach by comparing treated and untreated cells and normalized to the reference gene GAPDH.
Statistical analysis was performed using SPSS software to calculate significance (P < 0.05). All data are presented as mean ± standard deviation. The relative quantification diagram was performed using GraphPad Prism 8.0.1 software.
ABCC1 and ABCC2 transcription level
Figure 2 shows the transcription level of ABCC1 and ABCC2 in A549 lung cancer cell in response to either bixin or fucoxanthin, and those carotenoids in combination with cisplatin. It is showed that single treatment of bixin, fucoxanthin, or cisplatin down-regulated ABCC1 mRNA level. When fucoxanthin was combined with cisplatin, they are able to decrease the down-regulation of ABCC1. In the other hand, the expression level of ABCC2 from cells treated with cisplatin alone was greater down-regulated than cells treated with either bixin or fucoxanthin. When bixin or fucoxanthin was combined with cisplatin, they are able to increase the down-regulation of ABCC2.
Caspase 3 and p53 transcription level
Figure 3 shows that only bixin treated as single regimen upregulates caspase 3 and this expression was downregulated when cells was treated in combination with cisplatin. In addition, the present study suggests that both carotenoids, either alone or in combination with cisplatin, upregulated p53 gene expression.
ABC transporters are often overexpressed in both naive and post-treatment tumors and hamper chemotherapy by reducing the intracellular drug accumulation in targeted cancer cells. They are also involved in tumor biology by transporting tumor-enhancing molecules and/or in protein–protein interactions, thereby promoting cancer aggressiveness, progression, and patient prognosis. The number of ABC drug transporters identified has increased over time indicating that the MDR phenotype is considerably more complex and likely to be cell and tissue-specific. Thus, it is important to improve our understanding of the function of individual transporters, particularly at the transcriptional level in certain cancer cells in order to improve therapeutic strategies.
Previously, we reported that the administration of a certain ratio of the carotenoids, bixin, or fucoxanthin, in combination with cisplatin can increase the sensitivity of lung cancer cells to cisplatin, as these compounds act synergistically to inhibit cancer cell growth. Also, higher doses of fucoxanthin (IC75) reduced the required dose of cisplatin for the synergistic effect. The sensitivity of A549 cells was hypothesized to be associated with the inhibitory effect of carotenoids on the ABC transporter proteins responsible for the xenobiotic efflux, thereby increasing intracellular cisplatin concentrations and synergistic interactions and ultimately causing cell death. Therefore, the current study was conducted to assess the transcription of the ABC transporter family, ABCC1 and ABCC2 in lung cancer cells to understand how carotenoids alone and in combination with chemotherapeutic cisplatin regulate the transporters at the transcriptional level. In addition, the expression of cell death genes may also be associated with the response of the cells to DNA damage by the regulation of the ABCC1 and ABCC2.
The administration of bixin or fucoxanthin decreases the expression of ABCC1 and ABCC2, which is further significantly increased, especially ABCC1, when cells are treated with fucoxanthin in combination with cisplatin [Figure 2]. Yuan et al. (2022) recently proved that excess oxidative stress can repress the gene expression and activity of ABC transporters in A549 cells and at moderate ROS levels, the expression and activities of ABC transporters increase. Fucoxanthin not only possesses antioxidant activity but also can act as a pro-oxidant. The present study suggests that ROS generation due to the pro-oxidant activity of the carotenoids can repress the expression of ABCC1 and ABCC2. Furthermore, when combined with cisplatin, it is speculated that the antioxidant effect of carotenoids can partly protect cells from cisplatin-induced oxidative stress thereby increasing the expression of ABCC1, probably via the Nrf2 signaling pathway as carotenoids promote the Nrf2/ARE transcription system and signaling.[37–39] Higher dose of fucoxanthin compared to bixin may also influence the significant difference of ABCC1 expression when carotenoid was combined with cisplatin [Table 1].
Caspase 3 belongs to the cysteine protease family and acts as an effector enzyme in inducing apoptosis. The caspase-dependent apoptotic pathway consists of intrinsic and extrinsic pathways. The extrinsic pathway is caused by stimuli originating from outside the cell such as the binding of TNF to the receptors. For instance, Fasl binds to the fas receptor, causing the binding of the FADD protein, which then binds to pro-caspase 8 forming DISC (death-inducing signaling complex) and activating caspase 8 which activates caspase 3 or caspase 7 to execute apoptosis. The intrinsic apoptotic pathway occurs due to a stimulus that can change the structure of the mitochondrial membrane, leading to the release of cytochrome C which binds to Apaf-1 to form the apoptosome and activates caspase 9. Caspase 9 activates caspase 3 or caspase 7 to execute apoptosis. Our results suggest that only bixin treated as single compound upregulates caspase 3 and this expression was downregulated when cells were treated in combination with cisplatin [Figure 3]. The increased caspase 3 expression indicates increased apoptosis after bixin treatment. Whereas, the administration of fucoxanthin and cisplatin, either alone or in combination, may act through a caspase-independent pathway.
p53 is a transcription factor protein that induces apoptosis in response to DNA damage via the caspase-independent p53 pathway.[30,41] Activated p53 activates BH-3 proteins such as PUMA and NOXA which will further promote mitochondrial membrane permeabilization through the Bax-Bak channel, leading to the release of several proteins such as AIF (apoptosis-inducing factor), EndoG (endonuclease enzyme), and HtrA2/Omi, which translocate to the nucleus and stimulate caspase-independent apoptosis. The present study suggests that both carotenoids, either alone or in combination with cisplatin, upregulated p53 gene expression, indicating the mechanism of proliferation inhibition and apoptosis occurs via the p53 caspase-independent pathway.
Financial support and sponsorship
This research was funded by the Ministry of Research, Technology, and Higher Education, Indonesia under Research Grant No. 0647/III/LPPM.PM.10.02/6/2022.
Conflicts of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
ABC = ATP-Binding Cassette
ABCB1 = ATP binding cassette subfamily B member 1
ABCC1 = ATP binding cassette subfamily C member 1
ABCC2 = ATP binding cassette subfamily C member 2
ABCC3 = ATP binding cassette subfamily C member 3
ABCG2 = ATP binding cassette subfamily G member 2
ARE = Antioxidant Response Element
ATCC = American Type Culture Collection
DISC = Death-inducing signaling complex
EndoG = Endonuclease enzyme
GSH = Glutathione
IC = Inhibitory Concentration
MDR = Multidrug Resistance
MRP = Multidrug Resistant Protein
Nrf2 = Nuclear factor erythroid 2-related factor 2
PUMA = p53 upregulated modulator of apoptosis
PXR = Pregnane X Receptor
ROS = Reactive Oxygen Species
RT-qPCR = Real-Time Quantitative-Polymerase Chain Reaction
TNF = Tumor Necrosis Factor
This work was supported by the technical support of the Laboratory of Molecular Biology, School of Medicine and Health Sciences, and Laboratory of DNA Technology, Faculty of Technobiology.
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