Natural medicinal compounds target signal transduction pathways to overcome ABC drug efflux transporter-mediated multidrug resistance in cancer

Abstract

ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 are the major players in drug efflux-mediated multidrug resistance (MDR), which severely affects the efficacy of chemotherapy. Several synthetic compounds block the drug transport by ABC transporters; however, they exhibit a narrow therapeutic window, and produce side effects in non-target normal tissues. Conversely, the downregulation of the expression of ABC drug transporters seems to be a promising strategy to reverse MDR in cancer cells. Several signaling pathways, such as NF-κB, STAT3, Gli, NICD, YAP/TAZ, and Nrf2 upregulate the expression of ABC drug transporters in drug-resistant cancers. Recently, natural medicinal compounds have gained importance to overcome the ABC drug-efflux pump-mediated MDR in cancer. These compounds target transcription factors and the associated signal transduction pathways, thereby downregulating the expression of ABC transporters in drug-resistant cancer cells. Several potent natural compounds have been identified as lead candidates to synergistically enhance chemotherapeutic efficacy, and a few of them are already in clinical trials. Therefore, modulation of signal transduction pathways using natural medicinal compounds for the reversal of ABC drug transporter-mediated MDR in cancer is a novel approach for improving the efficiency of the existing chemotherapeutics. In this review, we discuss the modulatory role of natural medicinal compounds on cellular signaling pathways that regulate the expression of ABC transporters in drug-resistant cancer cells.

1. Introduction

Multidrug resistance (MDR) is a major obstacle in cancer treatment and disease management. MDR occurs due to diverse reasons, including various intrinsic and extrinsic factors (Gottesman, 2002; Mansoori et al., 2017). The factors contributing to MDR are overexpression of drug efflux systems, genetic and epigenetic modifications in cells, DNA repair mechanisms, the deregulated apoptosis process, tumor heterogeneity, the interaction of signaling pathways, and the cross-talk between cellular processes (Ambudkar et al., 1999; Vaidya et al., 2022; L. Zhang et al., 2023).

Membrane transporters are the specialized proteins that assist in the movement of ions, and small- and macro-molecules across biological membranes. Transporters include ATP-hydrolysis driven pumps, secondary transporters including solute carrier transporters (SLCs), and ion channels in the cell membrane responsible for the active movement of substances against the concentration gradient (Guan, 2022; Vasiliou et al., 2009). The ATP-binding cassette (ABC) transporters are the largest family of membrane transporters and the major cause of various diseases in humans including the development of MDR in cancer (J. Q. Wang et al., 2021). The ABC genes are divided into seven subfamilies (ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, and ABCG) depending on homology and structure similarity (Dean et al., 2022). Moreover, ABC transporters are structurally characterized by four domains, two cytoplasmic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). The NBD possesses the ATP-binding and hydrolysis site, and the TMD consists of a drug-binding pocket that recognizes and effluxes the substrates outside the cells (Huang and Ecker, 2023). ABC transporters such as P-glycoprotein (P-gp) or ABC subfamily B member 1 (ABCB1), Multidrug Resistance-Associated Protein-1 (MRP1) or ABC subfamily C member 1 (ABCC1), and Breast Cancer Resistance Protein (BRCP) or ABC subfamily G member 2 (ABCG2) are concurrently expressed in normal cells and overexpressed in many cancer subtypes (Giddings et al., 2021). Overexpression of these three ABC transporters plays a significant role in developing clinical MDR (Ambudkar et al., 2003; Robey et al., 2018). Multiple small-molecule inhibitors of the MDR-linked ABC transporters have been developed, but toxicity to normal cells limits their utility for cancer management (Abdallah et al., 2015; Lai et al., 2020).

Phytochemicals and their potent derivatives are considered as fourth generation P-gp inhibitors. Recent studies have shown the impact of phytochemicals on inhibiting P-gp-mediated drug transport. For example, a simplified tetrandrine derivative termed OY-101 efficiently inhibited P-gp-mediated drug transport and reversed drug resistance in Eca109 vincristine-resistant esophageal cancer cells (Zeng et al., 2023). Also, we have recently shown that an N-alkylated monoterpene indole alkaloid derivative, N-(para-bromobenzyl) tabernaemontanine (NBBT) synergistically improved the doxorubicin efficacy in the drug-resistant KB-ChR-8-5 cancer cell line (Bharathiraja et al., 2023). In another study, soliensinine, a bioactive alkaloid isolated from Nelumbo nucifera proved to be a potent chemosensitizer when combined with paclitaxel in MDR-HCT-15 colorectal cancer cells (Manogaran et al., 2023). Similarly, resveratrol was found to significantly improve the chemosensitization of 5-fluorouracil in drug-resistant colorectal cancer cells (Brockmueller et al., 2023). Moreover, secondary metabolites isolated from the endophytic fungus Pestalotiopsis microspore exhibited MDR reversal activity in adriamycin-resistant MCF7/ADR breast cancer cells (Qian et al., 2023).

Besides the inhibition of transporters, modulating different cellular signaling pathways responsible for ABC transporter gene expression is considered an effective strategy for the reversal of MDR. This review includes a discussion of the transcriptional factors (TFs) and signaling pathways involved in the regulation of ABC transporter mediated MDR in cancer cells as well as the importance of natural medicinal compounds that modulate the expression of ABC transporters via cellular signaling pathways to overcome chemoresistance in cancer.

2. Transcription factors (TFs) involved in ABC transporter mediated MDR

TFs are sequence-specific DNA binding proteins that are involved in cellular signal transduction pathways, thereby regulating gene expression in the cellular milieu. The TFs are pleiotropic and regulate various cellular signaling pathways. TFs are also responsible for the regulation of ABC transporters that are involved in the development of MDR, such as ABCB1, ABCG2, and ABCC1. Several TFs actively bind to the promoter region of ABC transporter genes and regulate their transcription. Table 1 shows different TFs involved in regulating the expression of MDR-linked ABC transporters and their binding sequences in the proximal promoter region.

Table 1.

List of TFs involved in regulating ABC transporter expression and their binding site in the proximal promoter region of MDR-linked ABC transporters

Transcriptional factor	ABC gene	Binding site in ABC gene promoter region	Gene expression pattern	Ref.
NF-κB	ABCB1	GGGAATTCTCCTTTCGGGG	Upregulation	(Chen et al., 2014)
AP-1	ABCB1	GCATTCAGTCAATCCG	Upregulation	(Chen et al., 2014)
ERRγ	ABCB1	TCAAGGTCA	Upregulation	(Chen et al., 2020)
NF-Y	ABCB1	CCAAT box	Upregulation	(Dolfini et al., 2016)
STAT3	ABCB1	TTCCACTAA	Upregulation	(Zhang et al., 2011)
Gli	ABCB1	GACCACCCA	Upregulation	(Chen et al., 2014)
CBF1	ABCC1	GTGGAGA	Upregulation	(Cho et al., 2011)
Androgen receptor (AR)	ABCG2	AAAGAAT	Upregulation	(Chung et al., 2019)
SMAR1	ABCG2	ATGCTGCA	Downregulation	(Xu et al., 2021)

2.1. Transcriptional regulation of MDR-linked ABC transporters

2.1.1. ABCB1 (P-gp)

ABCB1 is a well-characterized ABC drug transporter that is overexpressed in different types of cancers and facilitates the efflux of structurally dissimilar and amphipathic anticancer drugs. The human ABCB1 gene is positioned at chromosome 7q21.12 which encodes 170 kDa N-glycosylated P-gp (Fan et al., 2023). TFs such as Nuclear Factor of the κ-chain in B-cells (NF-κB), myc, AP-1, TNF-α, PTEN, Speciality protein 1 (Sp1), and Sp3 actively bind to the promoter region of the ABCB1 gene and modulate its expression (Lee and Thévenod, 2021). Recently, a study described the pleiotropic effects of a TF known as Basic helix-loop-helix family member e40 (BHLHE40), which regulates ABCB1 gene expression (Yin et al., 2023). BHLHE40 directly binds to the promoter region of ABCB1 (−1605 to −1597) and downregulates its expression in adriamycin-resistant chronic myeloid leukemia and breast cancer cells (Yin et al., 2023). Y-box binding protein 1 (YB-1), a TF encoded by the YBX1 gene, binds to exon 3 in the promoter region of the ABCB1 gene in the inverted CCAAT box site and upregulates the expression of P-gp (Taylor et al., 2023). Similarly, a study by Lu et al. demonstrated the role of hepatocyte nuclear factor 1 homeobox A (HNF1A) in regulating MDR-linked ABC transporter genes (Lu et al., 2019). HNF1A was revealed to be a transcriptional suppressor of the ABCB1 gene that binds to the upstream region of the ABCB1 promoter (−988 bp to −624) in PANC-1 pancreatic cancer cells (Lu et al., 2019). p53 is a tumor suppressor protein that directly binds to the proximal ABCB1 promoter region (−70 to −40) and represses its expression in drug-resistant cells, whereas mutated p53 strongly upregulates ABCB1 expression via ETS Proto-Oncogene 1 (Chin et al., 1992; Sampath et al., 2001; Scotto, 2003; Zhan et al., 2005).

2.1.2. ABCG2 (BCRP)

ABCG2 is a 70 kDa monomeric polypeptide encoded by the ABCG2 gene, located on chromosome 4q22.1 (Fan et al., 2023). The functional unit of ABCG2 is a homodimer, and it mediates the efflux of a number of cytotoxic drugs, leading to the development of chemoresistance. Key TFs such as CAR/PXR, PPAR, AHR, NRF-2, and Erα/β/PR-A/B can bind with particular DNA response elements, DR5 (−8000), PPRE (−3796), AHRE (−2333), AHRE (−1661), and ERE/PRE (−187) in the promoter region to activate ABCG2 transcription (Gorczyca and Aleksunes, 2020). Yang et al. discovered the Sp1 binding site at −212 base pair in the promoter region of ABCG2, and this site is essential for the maximal promoter activity in A549 cells (Yang et al., 2013). Nuclear erythroid 2-related factor 2 (Nrf2) directly binds to the antioxidant response elements (ARE) region (1431 to −420) of the ABCG2 promoter and upregulates its expression in the biliary tract and in gall bladder cancer cells (Zhan et al., 2019). Similarly, hypoxia-inducible factor-1 alpha (HIF-1α) directly binds to the hypoxia-response elements (HREs) found in the −290 to −101 region of the human ABCG2 promoter region to regulate BCRP expression in response to hypoxic conditions (Kazi et al., 2014).

2.1.3. ABCC1 (MRP1)

The ABCC1 gene is located on chromosome 16p13.11, encoding a 190 kDa protein. It is overexpressed in various cancer cells and confers drug resistance. TFs such as SRY-box transcriptional factor 2 (Sox2) and Sox18 upregulate ABCC1 gene expression in melanoma SP cells and brain microvascular endothelial (BMECs) cells (Si et al., 2020; Zhang et al., 2023). HNF1A also regulates ABCC1 gene expression in pancreatic cancer cells by binding to its promoter region (Lu et al., 2019). A homeodomain transcription factor known as Paired related to homeobox 1 (Prrx1) binds with the promoter region of the ABCC1 gene (−256 to −263) and mediates its expression in U251 and LN229 human glioma cells (Chen et al., 2022). The NOTCH1 TF was found to promote ABCC1 upregulation in MCF7/VP cells via intracellular C-terminal fragment (NCID) and CBF1 binding to the promoter region of the ABCC1 gene (+103 and −411) (Cho et al., 2011).

3. Natural medicinal compounds

Natural compounds are secondary metabolites with remarkable chemical diversity obtained from natural sources, such as plants (phytochemicals), microbes, and marine sources. They have historically been used in traditional medicine in raw form with known benefits and have been the basis of alternative medicine branches such as Ayurveda and Traditional Chinese Medicine (TCM). The increasing significance of natural compounds in the treatment of several diseases, including cancer, has led to the need for proper cataloguing and developing resources to search for these compounds. Natural compounds such as alkaloids, polyphenols, and terpenoids are the major classes that have the potential to modulate the expression of MDR-linked ABC transporters.

Several databases have been developed over the years, and they represent a large number of natural compounds, extracted from diverse sources. The largest freely available resource of natural compound collections is provided by the "Coconut" database (Collection of open natural products) (Sorokina et al., 2021), comprising >400,000 natural products with detailed annotation including name(s), source, structure, and any associated publication. The “natural product atlas”, is a collection of >32,500 unique compounds, primarily from microbial sources (Van Santen et al., 2019). These databases have helped to accelerate natural product discovery with the availability of information on purification and identification parameters. Supernatural 3.0 is another extensive database with >750,000 compounds and their isomers (Gallo et al., 2023). This database not only provides detailed annotation of compounds that are also linked to other databases, but also indicates their possible interaction partners or associated cellular pathways where the compounds can be used as inhibitors, and the purchase information. This database also provides a search engine for specific diseases such as compounds studied for specific cancer sub-types.

There are several advantages and disadvantages associated with natural product-based drug discovery (Table 2). Natural medicinal compounds possess unique benefits such as lower toxicity with better metabolic and systemic clearance. One example is the use of natural compounds to counteract cardiotoxicity. P-gp is known to be expressed in heart tissues (Melaine et al., 2002; Qu et al., 2022) and doxorubicin is a commonly used chemotherapeutic drug. Administration of doxorubicin with conventional P-gp inhibitors such verapamil, cyclosporine A, and tamoxifen causes cardiotoxicity by increasing doxorubicin accumulation in the heart (Hafez and Hassanein, 2022). Kim et al. discovered that the administration of phytochemicals such as piperine and capsaicin in male ICR mice models did not result in doxorubicin-mediated cardiotoxicity (Kim et al., 2018). Moreover, wogonin, a flavone, specifically inhibits P-gp in cancer cells but not normal cells, and induces apoptosis by increasing the intracellular retention of etoposide in cancer cells (Lee et al., 2009).

Table 2.

Advantages and disadvantages of using natural compounds as modulators of signaling pathways.

Advantages

Disadvantages

Natural compounds have specific targets, required for the inhibition of signaling pathways, which have cross-reactive steps Natural compounds can work synergistically without compromising the effect of other codrugs Natural compounds exhibit less toxicity and easy metabolism and clearance from the body Fewer side effects are observed with natural compounds, due to specific targets Being produced naturally, these compounds exhibit more chemical and physical stability Structural diversity and complexity Natural compounds offer an ease of usage as oral drugs, which is a preferred method for drug administration

Natural compounds are usually less potent and thus, are required in larger quantities It is difficult to screen and identify active components in pure form from the extract Due to the complexity of nature, natural compound structures can be of high molecular weight, difficult to solubilize, degrade faster, and are difficult to characterize In most cases, the functional mechanisms are unknown There is limited availability of source material Despite verified resources, there is an issue of dereplication and rediscovery of the same compounds, also leading to patenting issues Change in properties of active compounds from plants grown in different soil and environmental conditions

However, the identification and purification of clinically significant bioactive compounds from complex natural extracts or mixtures is a complicated and time-consuming process. Furthermore, poor bioavailability is a major challenge in the development of potent drug molecules from natural origins. These drawbacks could be overcome by recent nanotechnology-based pharmaceutics approaches. The nano-based carriers play a significant role in addressing the drawbacks of natural medicinal compounds by enhancing their bioavailability, therapeutic efficacy, and controlled release in the targeted cellular milieu (Mitchell et al., 2021; Patra et al., 2018; Yadav et al., 2022). Table 2 enlists the major advantages and disadvantages of using natural compounds.

3.1. Natural medicinal compounds in clinical trials

Several natural medicinal compounds are under clinical trials concerning cancer disease management. A phase-1 clinical study demonstrates the potential of curcumin in combination with an EGFR-TKI in NSCLC patients (Esfahani et al., 2019). Moreover, treatment with curcumin reduces NF-κB DNA binding ability in breast cancer patients (NCT01740323). A phase 2 clinical trial demonstrated that lycopene suppresses Akt/GSK3β/β-catenin signaling and hippo pathways, and has a positive effect in reducing anti-EGFR drug toxicity (Moroni et al., 2021). Resveratrol decreased gastrointestinal neuroendocrine tumor progression via activating Notch-1 signaling (NCT01476592). Furthermore, resveratrol significantly inhibited the Wnt signaling pathway in colon cancer patients (Holcombe et al., 2009). A phase 2 randomized double-blinded clinical study (NCT04597359) showed the role of green tea catechins in the prevention of prostate cancer progression. Epigallocatechin, a phytochemical found in green tea extracts is under interventional clinical trial in colorectal cancer patients (NCT02891538). PSC833, also known as valspodar, is a derivative of cyclosporine A which is produced by the aerobic fungi Trichoderma polysporum and Tolypocladium inflatum. A phase 2 clinical trial combining PSC833 and paclitaxel showed a decrease in metastatic and recurrent breast tumors and reversed drug resistance (NCT00002826). A phase 2 randomised, double-blinded, placebo-controlled trial was conducted to investigate the effectiveness of paclitaxel and curcumin treatment when given once a week for 12 weeks to patients with advanced and metastatic breast cancer (NCT03072992).

3.2. Natural phytochemicals modulate the expression of MDR-linked ABC transporters via cellular signaling pathways

The expression of MDR-linked ABC transporters is regulated by various TFs. Signal transduction pathways such as JAK/STAT, PI3k/Akt, NF-κB, MAPK/ERK, Notch, Wnt/β-catenin, Hedgehog, Hippo/YAP, NRF2, and HIF-1α play a crucial role in the development of MDR in cancer (Figure 1) (Gao et al., 2021; Kumar et al., 2021; Liu et al., 2020; Trojani et al., 2019; Yang et al., 2022). Certain phytochemicals are able to modulate cell signaling pathways in several types of cancers (Garg et al., 2023). Studies have demonstrated that many phytochemicals work in synergy with established chemotherapeutic drugs. Moreover, phytochemicals selectively target the signal transduction pathways in cancer cells and facilitate the effectiveness of chemotherapeutic drugs (Fernando et al., 2019; Hadi et al., 2000; Prasad et al., 2011).

Figure 1. Signal transduction pathways related to ABC transporters (ABCB1, ABCG2, and ABCC1):

Schematic illustration showing signaling pathways and their transcription factors (TFs) regulating gene expression of ABC transporters. TFs such as STAT3, Gli, YAP/TAZ, NRF2, NF-κB, β-catenin/TCF, Csl, and ERK 1/2 play a pivotal role in the signaling pathways that activate MDR-linked ABC transporter expression during the development of drug-resistant cancer. After transcription, ABC transporter proteins are translocated to the endoplasmic reticulum for folding and post-translational modifications (PTM). ABC transporter proteins are glycosylated in the Golgi apparatus and trafficked to the cell membrane. Finally, the ABC transporter proteins are translocated to the plasma membrane and are involved in the drug-efflux function.

Natural phytochemicals modulate the activation of TFs and thereby regulate the expression of ABC transporters (Choudhari et al., 2020; Martins-Gomes and Silva, 2023; Teng et al., 2021; Zunica et al., 2022). For example, as discussed in previous sections, curcumin is a Generally Recognized As Safe (GRAS)-approved phytochemical, which downregulates the expression of ABC drug transporters and sensitizes cancer cells to chemotherapeutics (Shaikh et al., 2021). Several marine and microbial compounds were found to be efficient modulators of MDR-linked ABC transporters (Kumar and Jaitak, 2019). Iso-pencillixanthone A, a secondary metabolite of the marine fungi Penicillium oxalicum significantly reduced P-gp expression and reversed chemoresistance in vincristine-resistant HeLa cells and tumor xenograft models (Chen et al., 2018). Similarly, ecteinascidin 743, a natural marine tetrahydroisoquinoline alkaloid, downregulates ABCB1 gene expression in KB-C-2 cells ( Kanzaki et al., 2002). Table 3 lists various phytochemicals that act as potent modulators of MDR-linked ABC transporters in different cancer sub-types.

Table 3.

List of phytochemicals that modulate key cellular signaling pathways that affect the expression of ABC drug transporters

ABC transporter	Phytochemical	Experimental model	Mechanism of action	Ref.
ABCB1	Poncirin	Osteosarcoma MG63/CDDP and U2OS/CDDP cells	Downregulates ABCB1 expression by inhibiting PI3k/Akt pathway and reverses cisplatin resistance	(Zhao et al., 2021)
	4-hydroxyphenyl retinamide	Acute myeloid leukemic cells	Inhibits NF-κB and decreases ABCB1 expression	(Hui Zhang et al., 2020)
	Icaritin	Osteosarcoma MG-63/DOX cells	Inhibits STAT3 phosphorylation and decreases ABCB1 expression	(Wang et al., 2018)
	Fangchinoline	CEM/ADR5000	Decreases P-gp expression and increases Dox accumulation	(Sun and Wink, 2014)
	Tetrandine	Leukemic K562 cells	Decreases mRNA and protein expression of P-gp by inhibiting the NF-κB pathway	(Shen et al., 2010)
	Feroniellin A	Lung cancer A549RT-eto cells	Inhibits NF-κB and decreases P-gp expression	(Kaewpibo on et al., 2014)
	Oroxylin A	Breast cancer MCF7/ADR cells	Suppresses P-gp expression through Chk2/P53/ NF-κB pathway	(Zhu et al., 2013)
	Saikosaponin D	Breast cancer MCF7/ADR cells	Decreases mRNA and protein expression of P-gp	(Li et al., 2017)
	Ampelopsin	Leukemic K562/ADR cells	Decreases P-gp expression and enhances ADR accumulation	(Ye et al., 2009)
	Indole curcumin	MDR A549 cells	Downregulation of ABCB1 and COX 2 genes
ABCG2	Nuciferine	A549/T and HCT8/T	Inhibits PI3K/Akt/Nrf2 pathway and suppresses BCRP expression	(Liu et al., 2020)
	Ursolic acid	SKOV-sp	Downregulates ABCG2 and HIF-1α by inhibiting PI3K/Akt pathway	(Wang et al., 2016)
	Diflourinated curcumin analogue	Pancreatic AsPC-1 and MiaPaCa-2 cancer cells	Decreases ABCG2 expression	(Bao et al., 2012)
	Curcumin	Breast cancer MDA-MB-231 and MCF-7	Reduces ABCG2 expression and decreases breast cancer stem cell population	(Zhou et al., 2015)
	Poncirin	Osteosarcoma MG63/CDDP and U2OS/CDDP cells	Downregulates ABCG2expression by inhibiting PI3k/Akt	(Zhao et al., 2021)
	Guajadial	Breast cancer MCF-7/ADR and MCF-7/PTX cells	Inhibits the expression of ABCG2by suppressing PI3k/Akt pathway	(Li et al., 2019)
	Epigallocatechi n gallate	Breast cancer MCF-7TAM cells	Decreases ABCG2 expression	(Farabegoli et al., 2010)
	Resveratrol	PC9/G NSCLC cells	Combined with gefitinib decreasesABCG2 expression	(Zhu et al., 2015)
ABCC1	Berberine	MCF-7/DOXFluc	DecreasesABCC1 expression and enhances DOX accumulation	(Qian et al., 2021)
	Triptolide	Epidermoid cancer KB-7D, KB-tax cells and tumor xenograft	Decreases MRP1 protein expression and combined with 5-fluorouracil showed synergistic cytotoxic effects	(Chen et al., 2010)
	Tetrandrine	Esophageal squamous carcinoma YES-2/DDP cells	Decreases MRP1 expression and reverses cisplatin resistance	(Wang et al., 2012)
	Piperine	A-549/DDP	Decreases ABCC1 mRNA expressions and reverses doxorubicin resistance	(Li et al., 2011)
	Quercetin	HL-60/ADM, K562/ADM	Decreases ABCC1gene and MRP1 expression	(Cai et al., 2004)
	6-Gingerol, 10-Gingerol	Prostate cancer PC3 and PC3R	Decrease in MRP1 protein expression	(Liu et al., 2017)
	Daidzein	Breast cancer MCF-7 and MDA-MB-231 cells	Downregulate MRP1 protein expression	(Rigalli et al., 2019)
	Nobiletin	NSCLC A549/ADR cancer cells	Decreases MRP1 expression via modulating Akt/GSK3β/β-Catenin/MYCN signaling pathway and combined with ADR increased cytotoxicity in MDR cells	(Moon et al., 2018)

3.2.1. JAK/STAT pathway

The Janus Kinase (Jak)-signal transducer and activator of transcription (STAT) signaling pathway is one of the vital cellular pathways. The JAK/STAT pathway is a membrane-to-nucleus signaling module mediated by receptor tyrosine kinases that affects tissue repair, inflammation, immune fitness, haematopoiesis, and apoptosis (Hu et al., 2021). During the signaling process, when cells receive the signal, the receptor is dimerized and activates JAK proteins by transphosphorylation. STATs are then recruited to the plasma membrane and phosphorylated to form homo- and heterodimers. These complexes are subsequently translocated to the nucleus where they bind with DNA binding elements, which regulate the target genes (Chestnut et al., 2021; Fahmideh et al., 2022). Therefore, inhibiting the JAK/STAT pathway is a promising choice for cancer treatment. Studies have shown that phosphorylated STAT1 promotes P-gp overexpression and triggers drug resistance in patients receiving methotrexate (Nam et al., 2019; Qin et al., 2018).

STAT3-mediated drug resistance in tumors was shown to be reversed by methanolic extract of unripe fruits from Solanum nigrum, which actively inhibited JAK/STAT3 pathways and attenuated drug resistance in NCI/ADR-RES cells (Jagadeeshan et al., 2017). Curcumin, a diarylheptanoid compound, reverses gefitinib and erlotinib resistance in human NSCLC cells and acts as a receptor tyrosine kinase inhibitor (Chen et al., 2019). Plant-derived oils such as citral and extra virgin olive oil induce apoptosis in P-gp overexpressing glioblastoma cells, reversing MDR by suppressing the JAK/STAT pathway (Aparicio-Soto et al., 2016; Fahmideh et al., 2022). Zhang et al. demonstrated the role of Schisandrin A isolated from Fructus Schisandrae chinensis, a bioactive lignan used in traditional Chinese medicine, which reversed MDR in ABCB1 overexpressing MCF-7/Dox cells via STAT3 signaling (Zhang et al., 2018). Recently, Teng et al. found that rhein and its prodrug diacerein downregulate P-gp expression by inhibiting pSTAT3 activation (Teng et al., 2022).

3.2.2. NF-κB pathway

The NF-κB family consists of five members: RelA (p65), p50, c-Rel, RelB, and p52. They form homo or heterodimers to bind with the promoter regions of target genes (O’donnell et al., 2023). The p50 and p65 dimers are involved in the regulation of several genes. In basal conditions, the p50 and p65 dimers are inactive when bound to the IκB kinase and remain in the cytoplasm. Inflammatory signals can trigger the activation of IκB kinases (IKKα, IKKβ, and NEMO) that phosphorylate IκBβ and dissociate NF-κB p50 and p65 dimers, consequently translocating to the nucleus and regulating the expression pattern of various target genes (Shahbazi et al., 2020). The presence of an NF-κB binding site on the ABCB1 gene leads to its transcriptional upregulation in multidrug-resistant cancer cells. Previously, it has been reported that phytochemicals such as ferulic acid, curcumin, and voacamine significantly inhibit the nuclear translocation of NF-κB and thus reverse the overexpression of P-gp in the drug-resistant cancer cells (Lopes-Rodrigues et al., 2016; Muthusamy et al., 2019; Pellegrini et al., 2022). Furthermore, curcumin has been found to suppress NF-κB activation and sensitize chronic myeloid leukemic cells to imatinib (Bilajac et al., 2022). Similarly, hesperidin, a bioflavonoid, potentiates the action of imatinib in drug-resistant breast cancer cells by inactivating NF-κB signaling (El-Sisi et al., 2020). Evodiamine, a TCM compound, downregulates ABCG2 expression by inhibiting the p50/p65 NF-κB pathway in oxaliplatin-resistant HCT-1116/L-OHP colon cancer cells and induces apoptosis (Sui et al., 2016). Tetrandrine treatment prevents doxorubicin-mediated ABCB1 mRNA transcription by inhibiting NF-κB in drug-resistant osteosarcoma cells (Lu et al., 2017). Additionally, andrographolide significantly downregulates P-gp expression by inhibiting NF-κB translocation to the nucleus in drug-resistant KB-Ch^R-8-5 cells (Lakra et al., 2023).

3.2.3. Ras/Raf/MAPK pathway

The Ras/Raf/MAPK pathway is another important signal transduction pathway in cancer cells. Ras is a member of the small GTPase family activated by receptor tyrosine kinase (RTK). The binding of Ras kinase with GTP activates the Raf kinase, which then phosphorylates and activates MEK. Activated MEK triggers MAP kinase (MAPK), which regulates cell proliferation and cell survival (Degirmenci et al., 2020). The different isoforms of MAP kinases (ERK 1/2, JNKs, and p38) translocate to the nucleus and phosphorylate the TFs (Braicu et al., 2019). Ras/Raf/MAPK signaling regulates diverse cellular functions crucial for tumorigenesis. MEK activation has been responsible for acquired resistance in colorectal and NSCLC cells (Martinelli et al., 2017). Eum et al. showed that inhibition of Raf/MEK/ERK signaling downregulates ABCB1 expression, thereby reversing drug resistance in Ras-NIH 3T3/MDR cells (Eum et al., 2013). Various tyrosine kinase inhibitors including erlotinib, gefitinib, clolani, and middolin have been developed to overcome drug resistance but some cancer patients are resistant to these drugs (Yang et al., 2022). Fisetin, a flavonoid group of polyphenols, inhibits the activation of MAPK to sensitize erlotinib-resistant lung cancer cells to this drug (Zhang et al., 2016). Saponins isolated from Paris forrestii (Takht), a flowering plant native to China, significantly decreased the phosphorylated level of ERK, thereby downregulating P-gp expression in MCF-7/ADM cells (Chai et al., 2020). Similarly, a C21 steroidal glycoside, asclepiasterol, decreased the expression of P-gp by blocking the MAPK/ERK pathway (Yuan et al., 2016).

3.2.4. PI3K/Akt/mTOR pathway

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of the rapamycin (mTOR) signaling pathway are crucial for cell proliferation and survival. The PI3K/Akt/mTOR pathway contributes to the development of drug resistance by activating ABC transporter overexpression in a variety of cancers such as breast, lung, and ovarian cancer, and in leukemia, which may enhance drug efflux (Liu et al., 2020). The PI3K pathway affects lipid kinases, which are activated by binding to growth factor receptors such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR), by employing an adaptor protein which binds with p110 and p85 to activate PI3K (He et al., 2021). The activated PI3K pathway converts phosphatidyl 3,4-bisphosphate (PIP2) into secondary messenger 3,4,5- triphosphate (PIP3). Activated PIP3 binds to phosphoinositide-dependent kinase-2 (PDK2), which phosphorylates and activates Akt kinase (Dong et al., 2021). Activated Akt kinase then translocates from the cytoplasm to the nucleus, which subsequently activates downstream genes such as mTOR and NF-κB. These proteins are actively involved in drug resistance by enhancing the expression of ABC transporters.

Although FDA-approved natural PI3K and Akt inhibitors attenuate tumorigenesis, they exhibit poor pharmacokinetic effects in clinical trials (Mishra et al., 2021). Phytochemicals including epigallocatechin-3 gallate, baicalin, resveratrol, osthole, and kanglaite are closely associated with the downregulation of ABCB1 through inhibiting the PI3K/Akt pathway (Ganesan et al., 2021). Liu and colleagues found that alkaloids such as nuciferine decrease the expression of ABCB1 and ABCG2, thereby overcoming the paclitaxel resistance in HCT-8/T and A549/T cells and tumor xenograft models by inhibiting the PI3K/Akt/ERK pathway (Liu et al., 2020). Chen et al. demonstrated that timosaponin A-III, a saponin extracted from A. asphodeloides decreased phosphorylated Akt levels and inhibited P-gp and MRP1 expression (Chen et al., 2016). Terpenoids such as guajadial, which is found in Psidium guajava, demonstrated antitumor activity by decreasing the expression of P-gp and ABCG2 transporters by suppressing the PI3K/Akt pathway in breast cancer cell lines (MCF-7/ADR and MCF-7/PTX) (Li et al., 2019).

3.2.5. Notch pathway

Notch signaling is a highly conserved ligand-receptor signaling pathway that regulates tumor stem cell formation and promotes drug resistance in cancer cells (BeLow and Osipo, 2020; Wang et al., 2010). There are four types of mammalian Notch receptors (Notch 1-4) that include canonical and non-canonical pathways. Canonical Notch signaling is initiated by ligand binding and then cleavage of notch receptor by γ-secretase, which releases the Notch intracellular cytoplasmic domain (NICD), subsequently translocating to the nucleus to target downstream genes (Guo et al., 2023). In contrast, the non-canonical Notch signaling is either dependent or independent of ligand interaction, modulating other signaling pathways including YY-1, NF-κB, β-Catenin, and HIF1α to indirectly alter the expression of ABCB1 (Lee and Thévenod, 2021). Anticancer drugs such as docetaxel and doxorubicin play a role in the Notch signaling pathway by increasing the expression of ABCB1 and MRP1, contributing to chemoresistance (Kumar et al., 2021). A recent clinical trial showed the effect of resveratrol on low-grade gastrointestinal neuroendocrine tumors via modulating Notch-1 signaling (Choudhari et al., 2020). In another study, resveratrol decreased the expression level of ABCB1 in HCT116/L-OHP colorectal cancer cells (Wang et al., 2015). Similarly, the di-fluorinated curcumin analog significantly decreased the expression of cleaved Notch-1 and ABCG2 levels in human AsPC-1 and MiaPaCa-2 pancreatic cancer cells (Bao et al., 2012).

3.2.6. Wnt/β-catenin pathway

The β-catenin pathway is a highly conserved signaling pathway. Catenin is the key member of the Wnt signaling pathway, responsible for tumor progression and drug resistance by activating various signaling factors. Normally, β-catenin on the cytoplasmic side of adherence junctions undergoes ubiquitination by proteasomal degradation. The Wnt ligand interactions inactivate GSK3β, which prevents the degradation of β-catenin, enhances its accumulation in the cytoplasm, and mediates its nuclear translocation (Paskeh et al., 2021). Then, β-catenin binds with the TCF-LEF family of TFs that interacts with the promoter region of genes (Schuijers et al., 2014). Lim et al. reported that hCMEC/D3 cells significantly overexpressed ABCG2 due to GSK3β inhibition and activation of β-catenin (Lim et al., 2008). It has been found that Lawsone (hennotannic acid) derivatives isolated from the henna plant inhibit Wnt signaling, which leads to the downregulation of ABCB1 and c-MYC (Hamdoun et al., 2017). Similarly, nobiletin, a citrus fruit phytochemical, inhibited the Akt/GSK3β/β-catenin/MYCN signaling pathway, thereby decreasing the overexpression of ABCC1 in NSCLC cells (Moon et al., 2018). Furthermore, the phosphorylation of Akt and GSK3β and changes in the level of β-catenin targeting MYCN downregulation resulted in the reversal of drug resistance in NSCLC cells. A study by Chen et al. showed that quercetin suppressed ABCB1, ABCC1, and ABCC2 expression in BEL-7402 hepatocellular cancer cells by regulating FZD7/Wnt/β-catenin signaling pathway (Chen et al., 2018).

3.2.7. Hedgehog pathway

The Hedgehog (Hh) signaling pathway plays an important role in embryogenesis and is active in most solid tumors. It is mostly silenced in adult tissues and abnormal activation can lead to cancer progression. Canonical Hh signaling is initiated by binding of ligands to Patched 1 (PTCH 1) and relieving Smoothened (SMO) receptor inhibition. SMO translocates to the primary cilia and activates the glioma-associated oncogene homolog (Gli), which travels to the nucleus to activate target gene expression (Skoda et al., 2018). Similarly, the non-canonical Hh pathway also regulates various target genes, with Gli activation occurring independently of SMO (Pietrobono et al., 2019). The G-protein-coupled receptor SMO has been recognized as a target for cancer chemotherapy by targeting the Hh pathway, and several inhibitors have been developed to inhibit SMO action. Sonidegib and vismodegib are two SMO inhibitors approved by FDA and other SMO inhibitors including saridegib and taladegib are in late phase clinical trials (Nguyen and Cho, 2022). Zhang et al. found that the ABCB1 gene is regulated by the HH signaling pathway in ovarian cancer cells (Zhang et al., 2020). The promoter regions of ABCB1 and ABCG2 possess a Gli binding site. Gli binds the GACCACCCA motif in the promoter region and regulates ABC transporter expression via the Hh signaling pathway in the ovarian and B-cell lymphoma cancer cells (Chen et al., 2014; Singh et al., 2011). Genistein, a flavonoid isolated from soybean potentially inhibits Hh-Gli signaling in prostate cancer cells (Zhang et al., 2012).

Furthermore, capsaicin, a proto-alkaloid isolated from capsicum, was reported to inhibit the Hh pathway, thereby inducing apoptosis and cell cycle arrest in nasopharyngeal carcinoma cells (Lin et al., 2017). The steroidal alkaloids isolated from Veratrum californicum have been reported to act as SMO inhibitors, including cyclopamine, which directly binds SMO and inhibits its activation (Seale and McDougal, 2022). The SMO inhibition indirectly prevents Gli translocation to the nucleus, which could be the reason for the downregulation of ABC transporter expression in drug-resistant cancer cells. The demethylated analog of cantharidin, a small molecule norcantharidin (NCTD) inhibited the Hh pathway to overcome P-gp mediated MDR in Dox-resistant (MCF-7R) human breast cancer cells (Chen et al., 2012).

3.2.8. Hippo/YAP pathway

The Hippo/yes-associated protein (YAP) signaling pathway was found to be a regulator of tissue growth in Drosophila and is highly conserved in mammals (Dong et al., 2007). The key components of Hippo pathways are neurofibromatosis type 2 (NF-2), mammalian STE20-like Ser/Thr kinases 1/2 (MST1/2), large tumor suppressor kinases 1/2 (LATS1/2), YAP, TAZ (paralog of YAP), MOB protein family member (MOB3A) and transcriptional enhancer factor domain family members 1-4 (TEAD 1-4) (Kodaka and Hata, 2015; Lee et al., 2022). When Hippo signaling is active, NF-2 phosphorylates MST1/2, which combines with SAV1, a Salvador family WW domain-containing protein to initiate LATS1/2 phosphorylation. Phosphorylated LATS1/2 further activates a cascade of reactions to phosphorylate YAP, which subsequently binds to the 14-3-3 protein and remains in the cytoplasm for proteasomal degradation (Hao et al., 2008). When the cell receives growth signals, MOB3A acts as a negative regulator of the Hippo pathway, which prevents the phosphorylation of MST1/2 and LATS1/2, thereby inhibiting YAP/TAZ phosphorylation (Dutchak et al., 2022). The dephosphorylated YAP/TAZ protein then translocates to the nucleus where it can bind with TEAD1-4 to regulate the expression of certain genes responsible for blocking apoptosis and the proliferation of cancer cells (Meng et al., 2016).

Hippo signaling plays a major role in chemoresistance in several types of cancer cells (Wang et al., 2023; Wang et al., 2023). The binding of active TFs YAP/TAZ to TEAD1-4 induce the upregulation of ABCB1, ABCG2, and ABCC1 genes in different types of cancer cells such as liver, ovarian and gastric cancer (Chen et al., 2018; Mohamed et al., 2018; Xia et al., 2014). The most commonly used chemotherapeutics in the clinic such as methotrexate and doxorubicin potentially reduce the cellular concentration of MST1 and LATS1/2 and mediate nuclear translocation of YAP to develop drug resistance in MG63 and U2OS osteosarcoma cells (Wang et al., 2016). This Hippo signaling-mediated acquired chemoresistance can be reversed by YAP1 inhibitors such as verteporfin and statins (Wei et al., 2023). Nevertheless, to date, these YAP inhibitors are not clinically used because of their pleiotropic effects and the requirement of high doses (Lee et al., 2022). Natural phytochemicals are reported as YAP inhibitors to overcome drug resistance mediated by Hippo signaling. Catechol, a secondary metabolite present in fruits and vegetables, was found to increase p-AMPK expression, thereby decreasing the levels of YAP downstream targets such as CTGF and CYR61 in Panc-1 pancreatic cancer cells (Moon et al., 2021). This YAP inhibition enhanced the chemosensitivity of gemcitabine in drug-resistant pancreatic cancer cells (Moon et al., 2021). Similarly, ursolic acid, a herbal compound, increased the levels of MST1/2 and LATS1, resulting in increased phosphorylated YAP expression in gastric cancer cells (Kim et al., 2019). Additionally, scutellarin, a natural flavonoid found in Scutellaria barbata, a herb used in traditional Chinese medicine, significantly increased MST1 and LATS1 expression and reduced YAP1, TAZ, cyclin D1, and c-Myc in colorectal cancer (Yang et al., 2022).

3.2.9. NRF2/KEAP1 pathway

Nrf2 is a well-known TF that mainly prevents cancer cells from oxidative damage and confers cell survival. Normally, Nrf2 is bound to the Kelch-like ECH-associated protein 1 (Keap1) and Cul3-RBx1-E3 ubiquitin ligase in the cytoplasm and undergoes proteasome-mediated degradation. When cells receive an oxidative stress signal, Nrf2 is dissociated from Keap1, translocates to the nucleus, and binds to the ARE region to regulate gene expression (Liu et al., 2020). The Akt pathway regulates Nrf2 by inhibiting GSK-3β. The binding of Nrf2 to ARE upregulates gene expression of detoxifying enzymes such as HO1 and NQ01, as well as MDR genes such as ABCB1, ABCC1, ABCC4, and ABCG2 (Li et al., 2018). The activated Nrf2 contributes to the development of resistance to chemotherapeutics. Mitoxantrone and doxorubicin cause elevated Nrf2 levels in MDA-MB-231 breast cancer cells associated with increased ABCB1 and Bcl-2 expression. Parthenolide, a natural compound extracted from Tanacetum parthenium, a flowering plant in the daisy family, significantly downregulates Nrf2 expression, thereby reversing the acquired drug resistance mediated by P-gp and Bcl-2 (Carlisi et al., 2017). Vielanin P, a meroterpenoid isolated from Xylopia vielana, an Asian evergreen tree, inhibits PI3K/Nrf2 signaling pathway, thereby suppressing the mRNA and protein expression of ABCC1 in MCF-7/ADR and K562/ADR cells (Gao et al., 2019). Wang et al. demonstrated that NF-κB/p65 transcriptionally activates Nrf2 by binding with the κB2 site in the Nrf2 promoter region and significantly enhances the expression of ABCC2. Interestingly, dihydromyricetin, a flavonoid from Vitis heyneana, a climbing vine in the grape family, inhibits the p65 subunit of NF-κB from binding with the promoter region of Nrf2 and thus reduces the transcription and nuclear translocation of Nrf2, which reverses ABCC1-mediated drug resistance in colorectal cancer cells (Wang et al., 2021). Recently, Sun et al. showed that hepatocellular tumor models exhibit resistance towards sorafenib through Nrf2 signaling and that alkaloid camptothecin downregulates Nrf2 levels, thereby sensitizing the hepatocellular tumors to sorafenib (Sun et al., 2023).

4. Hypoxia-inducible factor-1 (HIF-1) in ABC transporter-mediated drug resistance

Tumor formation leads to reduced oxygen and nutrient transport, causing a hypoxic condition. The HIF-1 family regulates cellular signaling in response to hypoxia and allows cancer cells to proliferate in a hypoxic microenvironment. It consists of two subunits namely HIF-1α (oxygen-sensitive subunit) and HIF-1β (constitutively expressed) (Semenza, 2012). In normal oxygen level conditions, HIF-1α undergoes hydroxylation and subsequently interacts with pVHL (von Hippel-Lindau tumor suppressor) to promote ubiquitin-mediated degradation in proteosomes. But under hypoxic conditions, HIF-1α degradation is prevented, and it interacts with the HIF-1β to form a transcriptional dimer by basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) motifs, then accumulates in the nucleus and binds to the HRE to upregulate various target genes (Jaakkola et al., 2001; Masoud and Li, 2015; Wu et al., 2015). Hypoxia has been shown to activate signal transduction pathways such as PI3K, Nrf2, Notch, mTOR, NF-κB, and the ERK pathway, which subsequently promote nuclear accumulation of HIF-1α and mediate expression of target genes (Luo et al., 2022). Overexpression of HIF-1α contributes to ABC transporter-mediated drug resistance in cancer cells during hypoxia (Cosse and Michiels, 2008; Yong et al., 2022). The expression of ABCB1 was found to be increased in cycling hypoxia conditions in glioblastoma cells (Chou et al., 2012). HIF-1α induces ABCB1 expression in MDA-MB-231 cells (K. Wang et al., 2018). Interestingly, HIF-1α transcriptionally regulates the YY1 transcription factor, which in turn increases the expression of ABCB1 in leukemic cell lines (Antonio-Andrés et al., 2018). Krishnamoorthy et al. showed the link between ABCG2 expression and hypoxia and found HRE in the proximal promoter region of ABCG2 (Krishnamurthy et al., 2004; Krishnamurthy and Schuetz, 2006). Hypoxia induces ERK1/2 phosphorylation, which activates and accumulates HIF-1 in the nucleus, thereby upregulating ABCG2 in pancreatic cancer cells and resulting in resistance to gemcitabine (He et al., 2016). In one study, a luciferase reporter assay and chromatin immunoprecipitation analysis confirmed that the ABCC1 gene promoter contains an HRE region at −378 to −373, where the HIF-1α binds and transcriptionally regulates the ABCC1 gene in colon cancer cells under hypoxia (Lv et al., 2015). Various bioactive phytochemicals inhibit HIF-1α signaling to overcome cancer drug resistance mediated by ABC transporters under hypoxic conditions (Figure 2).

Figure 2. Pathways associated with the hypoxia-induced expression of MDR-linked ABC drug exporters.

Signaling modules affected by hypoxic conditions in tumor cells are shown. Selected natural phytochemicals are highlighted that inhibit the transcription factors involved in various signaling pathways to regulate MDR under hypoxic conditions.

Oroxylin A, a bioactive flavonoid, directly binds to the bHLH-PAS domain and inhibits HIF-1α binding with HRE3 in XPC promoter to reverse hypoxia-induced cisplatin resistance in NSCLC cells (Liu et al., 2020). Increased expression of Nrf2 and HIF-1α is responsible for the upregulation of ABCB1 and ABCG2 genes, which leads to chemoresistance (Vega et al., 2018). Nuciferine, an alkaloid, suppresses the activation of Nrf2 and HIF-1α, thereby reducing ABCB1 and ABCG2-mediated resistance to paclitaxel in HCT-8/T and A549/T cancer cells (Liu et al., 2020). Similarly, ursolic acid inhibits PI3k/Akt signaling under hypoxic conditions and downregulates HIF-1α and ABCG2 expression in ovarian cancer stem cells (W. J. Wang et al., 2016). Berberine, a benzyl isoquinoline has been shown to reverse doxorubicin resistance in breast cancer cells by downregulating ABCB1 and ABCC1 overexpression in MCF-7/DOX^Fluc and MCF7/ hypoxia cells and lowering levels of HIF-1α and AMP-activated protein kinase (Pan et al., 2017; Qian et al., 2021).

Certain phytochemicals are known to promote proteasomal degradation of HIF-1α, which may be able to reverse HIF-1-mediated cancer drug resistance. For example, degulin, a rotenone derivative, disrupts the function of heat shock protein 90 (HSP90) by binding to the ATP- binding pocket of chaperone, thereby promoting the proteasomal degradation of HIF-1α in H1299 tumor xenografts (Oh et al., 2007). Chrysin, a flavone present in honey, was shown to elevate HIF-1α prolyl-hydroxylation, which led to ubiquitination and degradation of HIF-1α in DU145 human prostate cancer cells (Fu et al., 2007). In breast cancer cells, EGCG, a polyphenolic compound found in green tea, significantly reduced the production of lactate dehydrogenase, resulting in proteasomal degradation of HIF-1α (Wang et al., 2013). Another natural compound, STAT3, was found to regulate the stabilization of HIF-1α by inhibiting its interactions with VHL (Jung et al., 2008). Sanguinarine, an alkaloid, was shown to inhibit the interaction of STAT3 and HIF-1α under hypoxic conditions, subsequently leading to proteasomal degradation in breast cancer cells (Su et al., 2021).

5. Gene regulation by histone deacetylases (HDACs) and ABC drug efflux pumps

Nucleosomes are the packaged units of chromosomal DNA wrapped around a core of eight histone proteins. Post-translational modifications, including acetylation, phosphorylation, and methylation of histones significantly affect gene expression via altering chromatin structure (Han et al., 2019). Reversible histone acetylation is modulated by two antagonist enzymes i.e., histone acetyltransferases (HATs) and histone deacetylases (HDACs). The activities of HDACs are regulated by several cellular functions such as subcellular localization of metabolic factors, post-translational modifications, protein- protein interactions, and carcinogenic events (Wang et al., 2022).

Pacheco et al. investigated the combinational effect of hydrazine and HDAC inhibitors—panobinostat and valproic acid, in three prostate cancer cell lines (DU145, PC-3, LNCaP) and in stromal cells (WPMY-1). This combinational treatment of hydrazine with HDAC inhibitors was shown to control cell proliferation and induce apoptosis (Pacheco et al., 2021). Yin et al. studied the synergistic effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) with the PARP inhibitor veliparib in prostate cancer. This study showed a reduction of BRCA1 protein levels via targeting the UHRF1/BRCA1 protein complex, which leads to increased DNA damage in cancer cells (Yin et al., 2018).

Although HDAC inhibitors represent promising pharmacological drugs to treat cancer, they have also been shown to significantly contribute to the development of drug-resistant cancer. Enhanced HDAC activity is associated with abnormal cellular proliferation and metastasis. Romidepsin (also known as depsipeptide or FR-901228), a natural product obtained from the bacterium Chromobacterium violaceum, downregulates the expression of the ABCG2 gene in MCF-7 and SF295 cells via the HDAC pathway. It was also found to significantly upregulate the mRNA and protein expression of ABCG2 in renal and colon carcinoma (To et al., 2008). Romidepsin is known to be an excellent P-gp substrate, resulting in enhanced expression of the efflux transporter in T-cell lymphoma patients (Gottesman et al., 2002). In renal carcinoma patients, the administration of romidepsin increases the level of ABCB1 and ABCG2 gene expression in malignant peripheral blood mononuclear cells (Robey et al., 2006). Sodium phenylbutyrate (PBA), another HDAC inhibitor, induces the expression of ABCB1 and ABCG2 in acute myeloid leukemic (AML) cells, thereby contributing to drug resistance (Hauswald et al., 2009).

Wang and co-workers demonstrated that HDAC inhibitors such as SAHA, also known as vorinostat, and trichostatin A (TSA) considerably enhance the expression of ABCB1 at the mRNA and protein level, but have no effect on the protein expression of ABCG2 (Wang et al., 2019). Vorinostat was also found to downregulate the expression of ABCB1 in doxorubicin-resistant neuroblastoma [SK-N-SH and SK-N-Be(2)C] cell lines (Lautz et al., 2012). TSA, which is isolated from Streptomyces potentially induces p21WAF1 gene expression in cells and downregulates ABCC2 expression in KBV20C cells (Kim et al., 2011). Furthermore, TSA was found to significantly downregulate the expression of ABCB1 and ABCC1 genes in H69VP drug-resistant cells (El-Khoury et al., 2007). TSA was shown to bind to the promoter region of the ABCB1 gene and thereby inhibit the expression of P-gp in the K-562 d450 resistant cell line (Balaguer et al., 2012).

The short-chain fatty acids (SCFAs) including acetate, propionate, and butyrate are major metabolites produced by the gut microbiota that serve as HDAC inhibitors. Butyrate downregulates the expression of the ABCB1 gene and phosphorylates p65 levels via inhibiting the HDAC/NF-κB pathway in Caco-2 cells (Xie et al., 2021). Phytochemicals have the potential to modulate the activity of HDACs, thereby overcoming drug resistance mediated by ABC drug transporters. Baicalein, a natural flavonoid present in Scutellariae radix, the dried root of a medicinal herb, showed significant inhibition of HDACs 1 and 8 by degrading the HDAC-1 via the ubiquitin-proteosome pathway, further suppressing the growth of AML cells. Interestingly, baicalein upregulates the acetylation of histone H3 by degrading HDAC-1 and also inhibits the ABC transporter gene expression (Li et al., 2011; Yu et al., 2020).

Resveratrol treatment and knockdown of metastasis-associated protein 1 (MTA1)/HDAC unit increase the PTEN levels that trigger a decrease in p-Akt expression and proliferation index in prostate cancer (Dhar et al., 2015). Venturelli and co-workers demonstrated the role of resveratrol in the significant inhibition of HDACs and hyperacetylation of histones in hepatoblastoma cells (Venturelli et al., 2013). One study demonstrates that curcumin re-establishes the equilibrium between histone acetyltransferase and histone deacetylase activity, thereby selectively modulating the expression of genes that are implemented in cancer death (Teiten et al., 2013). Lycorine significantly inhibits the activity of HDAC enzymes in CML (K562 cells) that eventually arrest the cell cycle and inhibit the growth of cells. It was evident that lycorine treatment significantly upregulates the p53 expression and its target gene product p21 (Li et al., 2012). Diallyl disulfide isolated from garlic was found to significantly modulate the expression of HDAC activity in Caco-2 cells. This natural compound induces the upregulation of p21 (was1/cpi1) expression, inhibiting HDAC activity and histone hyperacetylation, thereby inhibiting cell proliferation (Druesne et al., 2004). The tropolone derivative β-Thujaplicin significantly inhibits the HDAC enzyme activity and inhibits the growth of T-lymphocytes (Ononye et al., 2013).

6. Regulation of intracellular localization of drug transporters by natural products

Various cellular signaling mechanisms are involved in the regulation of trafficking of mature transporter proteins to the cell membrane and intracellular compartments from the site of synthesis (Wakabayashi et al., 2006). Proteins that are misfolded are kept in the endoplasmic reticulum (ER) before being degraded. In order to be further processed and covalently modified, properly folded proteins are moved from the ER to the Golgi apparatus. They are then packaged into secretory vesicles and moved to the plasma membrane (Brouwer et al., 2022). N-linked glycosylation regulates membrane targeting, protects proteins from proteolysis, and ensures proper folding of the drug transporters. Several N-glycosylation inhibitors were discovered to prevent the development of MDR by regulating the internalization and localization of drug transporters. Tunicamycin, a naturally occurring antibiotic derived from Streptomyces lysosuperificus inhibits the transfer of UDP-N-acetylglucosamine (GlcNAc) to dolichol phosphate, preventing N-glycosylation and reducing protein maturation in eukaryotes (Hakulinen et al., 2017; Yoo et al., 2018). Additionally, Hou et al. showed that tunicamycin drastically decreased ABCG2 overexpression by altering its subcellular localization, reversing cisplatin resistance in hepatocellular carcinoma (Hou et al., 2013). Swainsonine, a compound isolated from the legume Astragalus lentiginosus, was reported to be an inhibitor of N-glycosylation in P-gp, thus downregulating its expression and reversing cisplatin resistance in Ehrlich ascites carcinoma (Santos et al., 2011). The cellular fate of P-gp in steady state was initially described by Katayama et al., proving that P-gp is degraded after being internalised in lysosomes (Katayama et al., 2015). Identification of natural compounds able to target lysosome-mediated P-gp degradation in drug-resistant cancer cells may pave the way to increasing chemotherapy efficacy in cancer patients.

7. Drug metabolism by MDR-linked cytochrome P450 (CYP) enzymes

The cytochrome P450 (CYP) enzymes play a crucial role in the detoxification of drugs and xenobiotics, and in cellular metabolism. They also facilitate pharmacokinetic drug-drug interactions (Liu et al., 2012). CYP1B1 is the most-studied CYP enzyme and is reported to be involved in cancer drug resistance (Yada et al., 2021). In tumor cells, CYP1B1 overexpression leads to a decrease in the efficacy of anticancer drugs and the development of drug resistance (Morvan et al., 2020). Due to their complex relationship, CYP and the ABC transporters play crucial roles in pharmacokinetics, pharmacodynamics and drug-drug interactions (Chan et al., 2023; Gillet et al., 2020). ABC transporters and CYP enzymes in tumour cells reduce the retention of chemotherapy drugs by either deactivating them or effluxing them from the cells (Hofman et al., 2019). The expression of CYP enzymes is regulated at various levels. Pregnant X receptor (PXR) is one of the main TFs regulating the gene expression of CYP enzymes (Gonzalez and Yu, 2006). PXR is a nuclear receptor superfamily involved in drug metabolism, removing toxic substances from tissues and clearing anticancer drugs from cells (Biswas et al., 2009; Mukherjee and Mani, 2010). Additionally, it was discovered that PXR activation increases the expression of MDR-linked ABC transporters, resulting in drug resistance (Chen and Nie, 2009; Niu et al., 2022). PXR exhibits a high tolerance towards chemotherapeutic drugs such as paclitaxel, cisplatin, docetaxel, tamoxifen, doxorubicin and sorafenib due to the regulation of MDR1/CYP3A4- mediated drug metabolism and the elevation of DNA damage repair, which together confer drug resistance (Chen et al., 2018; Feng et al., 2018; Niu et al., 2022; Qiao and Yang, 2014). According to the findings of Yu et al., some phytochemicals are non-specific P-gp inhibitors, which may have an impact on other proteins and enzymatic targets (Yu et al., 2016). For instance, quercetin can competitively inhibit MDR-linked ABC transporters and CYP3A4 enzyme activity to alter pharmacokinetic drug-drug interactions (Critchfield et al., 1994; Dewanjee et al., 2017).Makhov et al. investigated the effects of piperine in suppressing the activity of the CYP3A4 enzyme and further demonstrated that combining piperine and docetaxel effectively increased the pharmacokinetic activity and subsequently reversed MDR in castration-resistant prostate cancer cells (Makhov et al., 2012). Fucoxanthin, a marine-based carotenoid, was discovered to reduce rifampicin-activated CYP3A4 and P-gp overexpression through PXR-mediated signaling in HepG2 cells (Liu et al., 2012). Similarly, ginger extract was found to modulate the CYP1A2, CYP3A4 and P-gp expression in LS180 human colon adenocarcinoma cell lines (Brandin et al., 2007). In another study, baicalein, a flavonoid which is also an inhibitor of HDACs, was shown to increase the oral bioavailability of tamoxifen by preventing its CYP3A-mediated metabolism and P-gp efflux inhibition in the small intestine and in the liver (Li et al., 2011).

8. Conclusions and future prospective

The overexpression of ABC drug efflux transporters contributes to the emergence of MDR in cancer. Therefore, overcoming MDR by employing new and alternative strategies is one way to improve the efficiency of chemotherapy. Cellular signaling pathways are significantly involved in regulating the expression of MDR-linked ABC transporters. Natural medicinal compounds modulate MDR-linked ABC transporter expression, which might lead to the development of potential chemosensitizers. Phytochemicals such as curcumin, lycopene, and resveratrol regulate TF activation and modulate respective cell signaling pathways such as NF-κB, AKT/GSK3β/β-catenin and Wnt to inhibit the proliferation of drug-resistant cancer cells. The chemical scaffolds of these promising natural compounds can be utilized directly or to generate new derivatives for the development of potent drug molecules to treat drug-resistant cancers. Pre-clinical evaluation of either natural medicinal compounds alone or in combination with chemotherapeutic drugs is under rigorous investigation. The drawbacks associated with natural medicinal compounds, such as poor bioavailability, could be overcome by recent nanotechnology-based drug delivery technologies. Nano-based drug delivery methods facilitate increased solubility, selectivity, and bioavailability of natural compounds with the potential for targeted delivery. Therefore, natural medicinal compounds are promising modulators of ABC-transporter-mediated chemoresistance in cancer; however, additional clinical studies are required to check their precise mechanism of action, as well as their effectiveness in chemoresistant cancer sub-types. Furthermore, to prevent adverse toxicity during chemosensitization using natural medicinal compounds, it is necessary to extensively investigate the influence of natural compounds on the modulation of the expression of transporters at the blood-brain barrier and intestinal barrier. Moreover, the modulation of cytochrome P450, phase II conjugation enzymes and ABC transporters by natural medicinal compounds has been potentially linked to the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs. Therefore, studies on the interactions between natural medicinal compounds and chemotherapeutic drugs are essential to design the right dosage, timing, and formulation. Although a myriad of natural medicinal compounds can potentially reverse MDR in in vitro and in preclinical models, administering them to cancer patients in the clinic poses considerable challenges and warrants robust studies of drug-drug interactions. Therefore, collaboration among experts from different disciplines is needed to address and successfully employ prospective natural medicinal compounds as adjuvant therapy in combination with chemotherapy.

Highlights.

• ATP-binding cassette (ABC) transporters are the major players of multidrug resistance (MDR).

• Signaling pathways upregulate the expression of ABC drug transporters in drug-resistant cancers.

• Downregulation of the expression of ABC drug transporter reverses drug resistance in MDR cancer cells.

• Natural medicinal compounds downregulates the expression of ABC transporters in drug-resistant cancer cells via targeting signal transduction pathways.

• Therefore, natural medicinal compounds could be used as chemosensitizers to overcome ABC transporters mediated drug-resistance.

Abbreviations

ABC transporter

ATP-binding cassette transporter

AML

Acute myeloid leukemia

AHR

Aryl hydrocarbon receptor

ARE

Antioxidant response elements

AP-1

Activator protein 1

BHLHE40

Basic helix-loop-helix family member e40

BRCP

Breast Cancer Resistance Protein

CAR

Constitutive androstane receptor

CBF1

C promoter-binding factor 1

Coconut

Collection of open natural products

CYP

Cytochrome P450

EGFR

Epidermal growth factor receptor

Erα/β

Estrogen receptor alpha/beta

ER

Endoplasmic reticulum

ERK

Extracellular signal-regulated kinase

FGFR

fibroblast growth factor receptor

GlcNAc

N-acetylglucosamine

GLi

Glioma-associated oncogene homolog

GRAS

Generally recognised as safe

GSK-3β

Glycogen synthase kinase-3 beta

HATs

Histone acetyltransferases

HDACs

Histone deacetylase

HIF-1α

hypoxia-inducible factor-1 alpha

HNF1A

Hepatocyte nuclear factor 1 homeobox A

HRE

Hypoxia response elements

HSP90

Heat shock protein 90

JAK

Janus kinase

Keap1

Kelch-like ECH-associated protein 1

LATS1/2

Large tumor suppressor kinases 1/2

MAPK

mitogen-activated protein kinases

MDR

Multidrug resistance

MDR1

Multidrug Resistance Protein 1

MRP1

Multidrug Resistance-Associated Protein-1

MTA1

Metastasis-associated protein 1

mTOR

mammalian target of the rapamycin

MOB3A

MOB protein family member

MST1/2

Mammalian STE20-like Ser/Thr kinases 1/2

NBBT

N-(para-bromobenzyl) tabernaemontanine

NBD

Nucleotide-binding domains

NCTD

Norcantharidin

NF-2

Neurofibromatosis type 2

NF-κB

Nuclear Factor kappa B

NOTCH1

Neurogenic locus notch homolog protein 1

NICD

Notch intracellular cytoplasmic domain

NRF-2

Nuclear factor erythroid 2-related factor 2

PBA

Sodium phenylbutyrate

PDK2

phosphoinositide-dependent kinase-2

P-gp

P-glycoprotein

PI3K

Phosphoinositide-3 kinase

PIP2

phosphatidyl 3,4-bisphosphate

PIP3

phosphatidyl 3,4,5-triphosphate

Prrx1

Paired related homeobox 1

PPAR

Peroxisome proliferator-activated receptor

PTEN

Phosphate and tensin homolog

PTCH 1

Patched 1

PXR

Pregnane X receptor

RAF

Rapidly accelerated fibrosarcoma

RAS

Rat sarcoma

RTK

Receptor tyrosine kinase

SAHA

Suberoylanilide hydroxamic acid

SAV1

Salvador homolog protein 1

SCFAs

short-chain fatty acids

SLCs

solute carrier transporters

SMO

Smoothened receptor

Sp1

Speciality protein 1

Sp3

Speciality protein 3

Sox

SRY-box transcriptional factor

STAT

Signal transducer and activator of transcription

TCM

Traditional Chinese medicine

TEAD 1-4

Transcriptional enhancer factor domain family members 1-4

TFs

Transcription factors

TGF-β

Transforming growth factor-beta

TMD

Transmembrane domains

TNF-α

Tumor necrosis factor alpha

TSA

Trichostatin A

VEGFR

vascular endothelial growth factor receptor

WNT

Wingless related integration site

YAP

Yes-associated protein

YB-1

Y-box binding protein 1

YY-1

Yin Yang 1