Re-sensitization of cancer multidrug resistance through P-gp, MRP1 and BCRP modulation: advances in terpenoids based cancer therapy

Abstract

Among the most significant risks that many chemotherapeutic drugs currently encounter is the development of multidrug resistance (MDR) in cancerous cells. Numerous critical regulators are thought to be responsible for MDR and render therapeutic strategies inefficient. Several pathways mediate this effect, one of which is the upregulation of ATP-binding cassette (ABC) superfamily membrane transporters. Most of these transporters, which regarded as drug efflux pumps, are embedded in the cell membrane and include MDR-associated protein 1 (MRP1/ABCC1), MRP2, P-glycoprotein (MDR1/ABCB or P-gp), and breast cancer resistance protein (BCRP/ABCG2). The exploration of new potential modulators to diminish tumor MDR through natural product-derived compounds has emerged as a prominent research domain worldwide. Among different plant-derived natural compounds, terpenoids have recently attracted attention owing to their extensive health advantages. Multiple preclinical findings have primarily explored plant-derived terpenoids for their MDR-modulatory activities. Plant-based terpenoids counteract MDR by modulating signaling pathways or expression of pertinent proteins or genes. Thus, this review summarizes preclinical experimental data to elucidate the functional roles of terpenoids in MDR reversal by targeting ABC transporters and sheds light on future research on terpenoid-based cancer therapy.

Introduction

Currently, cancer poses an important global public health concern. The inability of some cells to undergo cell death and subsequent unchecked cell proliferation are the underlying causes of this disease [1]. In the most recent predictions for the year 2020, there were approximately 19.3 million new instances and approximately 10 million fatalities attributed to this ailment. Furthermore, these projections will unlikely improve in the forthcoming decades, as an estimated 30.2 million cases are anticipated by 2040 [2]. Chemotherapy is a highly recommended first-line treatment for this disease. Nevertheless, it is important to note that primary therapy resistance is an individual issue that contributes to the limited effectiveness of treatment [3]. Conversely, tumors may develop acquired resistance to primary chemotherapy, even if they initially respond to treatment [4, 5]. In addition, malignant cells may develop multidrug resistance (MDR), a condition in which they not only become resistant to the anticancer drug being used but also acquire cross-resistance to a wide range of molecules with dissimilar structures and mechanisms of action. The various factors that contributing to MDR are intricate and multifaceted [6]. MDR in cancer cells develops for a variety of reasons, some of which are genetic, whereas others are mediated by miRNAs and lncRNAs [7]. MDR genes play a pivotal role in drug resistance. Researchers have explored four genes that belong to the multidrug resistance family, two of which, MDR1 and MDR2, are expressed in humans. MDR1 encodes P-glycoprotein, an efflux pump that is dependent on calcium and has been linked to the emergence of resistance to anthracyclines, actinomycin D, paclitaxel, and vinca alkaloids [8, 9]. However, MDR2, commonly referred to as ATP-binding cassette subfamily B member 1, is not directly linked to multidrug resistance, and is mainly implicated in phospholipid transport. In a gene transfer experiment, MDR cDNAs were used to determine the effects of eukaryotic promoter-controlled elevated P-gp expression in cultivated cells that had previously been responsive to chemotherapeutic drugs [10, 11].

Resistance of cancer cells to existing cancer treatments is one of the main reasons why cancer treatments fail, which in turn causes cancer metastasis and recurrence (Fig. 1.). Malignant tumors are resistant and difficult to treat because of acquired resistance from prolonged exposure to cancer treatments and intrinsic resistance originating from the genetic features of cancer cells [12]. Moreover, significant adverse effects and high costs of existing cancer treatments impede their clinical utilization [13]. Consequently, the urgent development of innovative anticancer pharmaceuticals or therapies is essential.

Fig. 1.

Schematic representation of multidrug resistance in cancer by efflux transporters (P-gp, BCRP and MRP1) and their role in carcinogenesis process

Plant-derived natural compounds have been extensively studied for their antitumor potential to combat multidrug resistance in cancer. These compounds can be categorized into various classes such as polyphenols, flavonoids, terpenoids, alkaloids, and coumarins [14]. One of the classes of natural compounds, terpenoids, displays multiple anticancer actions, including the modification of drug-metabolizing enzymes and inhibition of cancer cell growth, hence adding to the ability to overcome treatment resistance [15]. Research into identifying MDR modulators is gaining interest among contemporary researchers due to their low toxicity and high specificity. Therefore, the main goal of this study was to provide a thorough overview of diverse plant-derived terpenoids as MDR modulators in distinct carcinomas, along with details about their structures and modes of action. Furthermore, we examined the principal targets associated with MDR in cancer and the anticarcinogenic effects of the terpenoids. This will provide primary information to medicinal chemists and scientists engaged in drug research, enabling the synthesis and identification of novel scaffolds with optimal efficacy in the future.

Multidrug resistance: efflux mechanism as potential target

The intricate relationship between cellular pathways and genetic alterations that cause cancer drug resistance has been elucidated in detail through recent developments in molecular biology. A significant area of advancement pertains to elucidating the molecular processes associated with the expulsion of drugs from cells [16]. Numerous studies have identified particular ATP-binding cassette (ABC) transporters, such as P-gp, MRP, and BCRP, which actively release drugs from cancer cells, thereby diminishing intracellular drug concentrations and resulting in MDR [17]. Owing to advancements in genomics and proteomics, it is now much simpler to detect genetic mutations and alterations linked to MDR. Resistance can develop when drugs interact differently or impact signaling pathways differently owing to alterations in genes targeted by drugs, such as tyrosine kinases or topoisomerases in cancer therapy [18].

Efflux mechanisms play crucial roles in the emergence of drug resistance in malignant cells. In these processes, anticancer medications are actively transported out of the cells, which lowers their intracellular concentrations and, in turn, their efficacy [19]. Drug efflux is primarily facilitated by ATP-binding cassette (ABC) transporters, including P-gp, MRPs, and BCRP. These transporters reduce drug buildup and promote drug resistance by pumping medicines out of cells using energy from ATP hydrolysis [20]. Several approaches and methods have been investigated to counteract these efflux mechanisms such as use of inhibitors, drug delivery systems and combination therapies [21]. Understanding the functions of P-gp, MRPs, and BCRP in mechanism of drug resistance is crucial for formulating therapeutic approaches to circumvent MDR in cancer [22, 23].

P-gp

The first protein linked to drug resistance to be identified was P-gp. The membrane glycoprotein P-gp, which has a molecular weight of around 170 kDa, was first discovered in Chinese hamster ovary cells (drug-resistant) in 1976 [24]. The most researched efflux transporter of ABC family is P-gp, which is encoded by ABCB1 [25]. Drugs used in chemotherapy that function as P-gp substrates are expelled from tumor cells [26]. ATP hydrolysis induces conformational alterations in the conformation of P-gp, facilitating the efflux of anticancer agents from the cell [27]. This efflux process largely entails the evacuation of substantial quantities of amphiphilic and nonionic compounds, including chemo drugs. The conformational alterations in P-gp are pivotal in the emergence of multidrug resistance in neoplastic cells and the diminished efficacy of chemotherapeutic agents [28]. Research has shown that P-gp overexpression causes chemotherapy resistance in several tumor types, including breast cancer, osteosarcoma, hepatocellular carcinoma, gastric cancer, and lung cancer [29–31]. Increased P-gp expression decreases intracellular drug levels and toxicity by facilitating the expulsion of anticancer drugs such as cisplatin, 5-fluorouracil, doxorubicin (DOX), and paclitaxel [32, 33]. This leads to insufficient inhibition of tumor cell proliferation by these drugs. Given the prevalent issue of clinical chemotherapy resistance, numerous research has investigated the mechanisms of P-gp-associated chemoresistance and the efficacy of P-glycoprotein inhibitors [34, 35]. Extensive research has been conducted on P-gp-targeted drugs, including competitive inhibitors such as cyclosporine A and non-competitive inhibitors like verapamil [36, 37]. These inhibitors increase the intracellular aggregation of chemotherapeutic drugs by blocking P-gp-mediated transfer of small molecules.

MRP1

Another ABC efflux transporter MRP1 was initially recognized in 1992 as the mechanism underlying MDR in the doxorubicin-selected human lung cancer H69AR cell line [38]. The MRP1 protein, with a molecular weight of 190 kDa and composed of 1531 amino acids, is expressed as ABCC1 gene [39, 40] and was the initial member found in the ABCC subfamily, which currently comprises 12 proteins [41–44]. In contrast to P-gp, human multidrug resistance proteins (MRPs) are capable of transporting organic anionic glucuronides, sulfates, and other compounds while also facilitating the excretion of diverse exogenous or endogenous compounds from the cell [45, 46]. The distinctive architecture of MRPs leads to divergent drug transport and retention patterns. In comparison to P-gp, which preferentially attached with substrates from the lipid bilayer, MRP1 can recruit its substrate directly from the cytoplasm [47]. According to recent research, the GSH synthase inhibitor (Buthionine Sulfoximine) can boost glutathione peroxidase function in human embryonic kidney cells, which in turn controls MRP1-mediated vincristine resistance [48]. These findings suggest that MRP1-mediated multidrug resistance may be facilitated by enhancing the efflux of glutathione-conjugated drugs from cancer cells.

BCRP

BCRP belongs to the ATP-binding transporter superfamily G, which is encoded by the ABCG2 gene localized on chromosome 4q22 [49, 50]. It is regarded as the most important component of MCF-7/MX cell drug resistance [51]. Human ABCG2 has a molecular weight of approximately 75 kDa and is a glycosylated plasma membrane protein. BCRP is typically located in the placenta, hematopoietic stem cells, liver, intestines, colon, lobules, and ducts of the breast [52, 53]. It can expel xenobiotics, organic drug conjugates, and endogenous substances through an ATP-activated efflux mechanism [54]. It encompasses a broad spectrum of substrates, such as hydrophobic chemotherapy drugs and hydrophilic organic anions conjugated with sulfate, glucuronate, and glutathione as the sulfated conjugates have superior compatibility with BCRP [55]. Furthermore, it can serve as a transport mechanism for nucleoside drugs [56]. Unlike most half-transporters, which tend to be found inside the cell, BCRP is located in the cell membrane [57, 58]. BCRP is expected to form various polymeric structural complexes via intramolecular disulfide interactions, and the development of an efflux channel aperture can markedly enhance its external pump efficacy. Only homodimers out of all polymeric structures are truly effective [59]. The energy-dependent export pump located within BCRP is a unique member of the ABC family [60]. A number of inhibitors, such as gefitinib, erlotinib, and lapatinib, have already been found to be successful in preventing BCRP-mediated resistance; some of these agents have already undergone clinical validation [61, 62].

Terpenoids as anticancer agent

Terpenoids extracted from plants have demonstrated potent efficacy against many cancer cell types in preclinical settings [63]. Tumor development is a complex process characterized by multiple hallmarks such as unchecked cell proliferation, apoptotic dysfunction, metastasis, and angiogenesis induction [64]. Terpenoids have anti-tumorigenic properties, indicating their potential application as chemotherapeutic drugs for tumor treatment [65]. Sesquiterpene lactones are implicated in the activation of apoptosis, tumor redifferentiation, and suppression of post-translational isoprenylation of proteins that regulate cell proliferation [66]. Helenalin has demonstrated significant efficacy in inducing apoptotic cell death in rhabdomyosarcoma (RMS) in vitro. Among children, RMS accounts for the vast majority of soft tissue sarcoma cases. The therapeutic benefits of helenalin are mediated by the formation of ROS, suppression of NF-κB p65 expression, induction of autophagy as indicated by enzymatic markers such as Atg12 and LC3-B, and activation of Caspase 3 and 9 cleavage [67]. Another terpenoid, limonene has been thoroughly evaluated in cancer cells, and its cytotoxic effects have been demonstrated in gastrointestinal cancer cells [68]. A study [69] recently showed that limonene enhanced its antiproliferative effects in melanoma and breast carcinoma cells via chitosan based nanoformulation. Borneol is a terpenoid with potential anticancer properties. It is an effective compound against various types of cancers, including esophageal squamous cell carcinoma, brain cancers, and melanoma [70–72]. Tetracyclic terpenes known as cucurbitacins, derived from Cucurbitaceae family plants, including cucumbers, gourds, and pumpkins, have anticancer properties [73]. Research has [74] demonstrated that cucurbitacin C therapy triggered G1 or G2/M cell cycle arrest in various cancer cell lines, while high-dose administration of this terpene resulted in apoptosis of cancer cells. Subsequent investigations demonstrated that cucurbitacin E increased DOX susceptibility against gastric cancer NCI-N87 cells [75], and that cucurbitacin D increased the chemotherapeutic potential of docetaxel in prostate cancer [76]. These studies demonstrated the potential of several terpenoids as anticancer agents and in increasing sensitivity to chemotherapeutic drugs [77]. Figure 2 demonstrated the chemical structures of terpenoids explored in the present study for their ability to reverse the MDR in various carcinomas (Fig. 2).

Fig. 2.

Chemical structures of plant terpenoids as modulators of ABC transporters in MDR

Mechanism of action of terpenoids in overcoming MDR in cancer

Numerous studies have indicated that plant-derived terpenoids serve as multifunctional medicines capable of addressing the primary reasons for multidrug resistance (MDR). Here, we describe the ways in which terpenoids, such as monoterpenoids, sesquiterpenoids, diterpenoids, and triterpenoids, can assist in decreasing MDR. Figure 3 summarized the mechanism of action of plant derived terpenoids in re-sensitizing the MDR.

Fig. 3.

Efficacy of plant terpenoids (monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids) in reversal of drug resistance via targeting ABC drug transporters such as P-gp, MRP1, BCRP in different preclinical cancer models

Efficacy of mono and sesquiterpenoids as re-sensitizing agent

Monoterpenoids and sesquiterpenoids have garnered significant interest as potential anticancer drugs. Many published articles have documented the antigenotoxic and chemopreventive properties of these terpenoids. Monoterpenoids and sesquiterpenoids exhibit significant multifunctionality, with their antitumor efficacy assessed by their capacity to induce apoptosis and mitochondrial dysfunction, inhibit cellular proliferation and angiogenesis, and alter multidrug resistance genes and proteins [78].

An in vitro study was conducted to evaluate the efficacy of natural substances against the multidrug-resistant HepG2 cell line (HepG2/ADM). Among many phytochemicals, the monoterpenoid cantharidin has demonstrated a notable ability to reverse multidrug resistance in liver cancer cells. These findings indicate that cantharidin markedly suppressed P-glycoprotein expression, mRNA transcription, and MDR1 promoter activity [79].

β-Elemene is a sesquiterpenoid phytochemical derived from the herb Curcuma rhizoma, utilized in traditional Chinese medicine for the treatment of several cancers, with no documented serious cytotoxic effects. Recent research, utilizing both in vitro and in vivo experimental settings alongside molecular techniques, has demonstrated that β-elemene can impede cell growth, halt the cell cycle, promote apoptosis, and reverse MDR [80].

A study reported that β-elemene (30 µmol/l) significantly enhances the susceptibility of breast cancer MCF-7/DOX cells to doxorubicin. Furthermore, the mechanisms by which β-elemene counteracts P-gp-mediated MDR indicated that β-elemene markedly enhances the intracellular concentrations of DOX and Rh123 by inhibiting the efflux action of P-gp in resistant MCF-7/DOX cells. β-elemene could suppress P-gp expression, but had minimal impact on MRP1 protein expression [81]. In a related study, Zhang et al. used a gene regulatory network to examine the impact of β-elemene on MDR in MCF-7 cells resistant to both BCA adriacin (Adr) and docetaxel (Doc). In both drug-resistant cell lines, β-elemene treatment considerably upregulated PTEN expression and significantly downregulated P-gp expression [82].

Artemisinin is a natural sesquiterpenoid lactone derived from Artemisia annua L., utilized as a traditional antipyretic agent for over two millennia in China. Artemisinin and its derivatives demonstrated anticancer efficacy against numerous tumor cell lines and animal models, with numerous clinical trials validating their potential as anticancer medicines [83]. Artesunate, a derivative of artemisinin, has shown potent anticancer properties in several cancer cell lines [84–86]. Another research demonstrated that artesunate markedly reversed drug resistance through P-glycoprotein downregulation in leukemia CEM/ADR5000 and CEM/VCR100 cells [87]. Additional studies have also shown the effectiveness of artesunate in reversing multidrug resistance in lung and esophageal cancers. There is evidence that artesunate decreased the growth of NSCLC A549 cells, triggered apoptosis, and reduced tumor growth in both in vitro and in vivo models. Moreover, artesunate decreased the expression levels of ABCG2, EGFR, and Akt at both mRNA and protein levels in these preclinical models [88]. A study revealed that artesunate contributed to overcoming drug resistance in esophageal cancer Eca109/ABCG2 cells. Artesunate successfully decreased drug resistance by decreasing ABCG2 expression and increasing the drug deposition of chemodrugs in cancer cells [89].

Parthenolide, another sesquiterpenoid, is extracted from the common medicinal plant feverfew and demonstrates a variety of biological effects, such as antimicrobial, anti-inflammatory, anti-leukemic, and anticancer properties [90]. Furthermore, parthenolide has demonstrated the potential to circumvent drug resistance in several carcinomas. A research reported that parthenolide mitigated DOX resistance in lung cancer A549 cells by diminishing NF-κB activity and HSP70 upregulation. Parthenolide suppressed P-gp upregulation and enhanced intracellular aggregation of DOX in A549/DOX cells. Parthenolide demonstrated a repressive effect on NF-κB stimulation in A549/DOX cells, indicating that suppression of NF-κB contributed to the reduction of P-gp expression. Furthermore, parthenolide efficiently inhibited elevated levels of HSP70 in DOX-resistant lung cancer cells. Overexpression of HSP70 enhanced P-gp level autonomously of NF-κB stimulation, while HSP70 knockdown resulted in decreased P-gp expression in these cancer cells. RT-PCR analyses indicated that HSP70 primarily regulates P-gp expression at the transcriptional level [91]. Research has shown that parthenolide exposure reversed MDR in the liver cancer cell line BEL-7402/5-FU. The results indicated that parthenolide exposure inhibited the proliferation and significantly enhanced the repressive effect of 5-fluorouracil drug against these drug resistant cells, thereby reversing the resistance of hepatocellular cancer cells. Additionally, parthenolide decreased NF-κB activity and P-gp, MRP, Bcl-2, and WNT1 expression, while enhancing p53 expression [92]. There is evidence that parthenolide could overcome drug resistance in leukemia K562/ADM cells. Parthenolide triggered cell death in K562/ADM cells and drug-resistant LSCs through a mitochondrial-mediated pathway. Additionally, parthenolide increased the sensitivity of K562/ADM cells to cell death in response to DOX by reducing P-gp function, which was mediated by the NF-κB signaling pathway [93]. Additionally, the drug resistance sensitization effects of parthenolide were investigated in breast carcinoma MDA-MB231 cells. The results revealed that parthenolide inhibited the Mitox and DOX linked drug resistance in MDA-MB231 cancer cells. This anticancer effect was accomplished by the suppression of both Nrf2 and its related target activities, including MnSOD, catalase, HSP70, Bcl-2, and P-gp [94]. Furthermore, a research found that parthenolide suppresses NF-κB activity in pancreatic cancer cell lines, thereby improving gemcitabine resistance. Parthenolide markedly enhanced growth inhibitory effects against gemcitabine-resistant and normal pancreatic cancer cells at doses of 10 µM and above, whereas NF-κB activity was dramatically suppressed even at 1 µM parthenolide. Moreover, western blot analysis revealed reduced MRP1 expression in gemcitabine-resistant pancreatic cancer cells exposed with a low dosage of parthenolide. Moreover, results of colony formation assay depicted that low parthenolide dosage enhanced the susceptibility of gemcitabine resistant pancreatic cancer cells [95].

Wilforine, a sesquiterpenoid, is a principal bioactive component derived from the woody vine, Tripterygium wilfordii. Hook. F. Wilforine has been utilized in the therapeutic management of rheumatoid arthritis, Crohn’s disease, HIV/AIDS, and different types of cancers [96]. A study assessed the regulatory effects of wilforine on P-gp expression and activity in cervical carcinoma HeLaS3 and KBvin cells. Wilforine markedly suppressed the P-glycoprotein efflux activity in a dose responsive manner. The subsequent kinetic study revealed that wilforine markedly decreased P-glycoprotein export function through competitive inhibition and enhanced the basal P-glycoprotein ATPase activity. Furthermore, wilforine re-sensitizes multidrug-resistant tumor cells to chemotherapeutic agents. The docking model demonstrated that wilforine was associated with P-glycoprotein residues, including LEU884, LYS887, THR176, and ASN172 [97].

Similarly, another study examined the efficacy of sesquiterpene tenulin and isotenulin on P-gp in cervical carcinoma HeLaS3 and KBvin cells. Tenulin and isotenulin markedly suppressed P-glycoprotein efflux by enhancing the P-glycoprotein ATPase activity. Tenulin and isotenulin interact with the efflux of rhodamine 123 dye and DOX via competitive and noncompetitive mechanisms, respectively. The simultaneous administration of tenulin and isotenulin with chemotherapy drugs markedly re-sensitized multidrug-resistant cancer cells. Additionally, tenulin and isotenulin demonstrated synergistic apoptotic effects with vincristine in a human multidrug-resistant cervical cancer cell line [98]. We have summarized the anticancer mode of action of mono and sesquiterpenoids in several carcinomas via downregulating P-gp, MRP1 and BCRP in Table 1.

Table 1.

Mono and sesquiterpenoids as resensitizing agent via P-gp, MRP1 and BCRP modulation

Mono and sesquiterpenoid	Cancer	Model	Target of drug resistance	References
Cantharidin	Hepatocellular carcinoma	HepG2/ADM cells	↓ P‑gp, MDR1 promoter activity	[79]
Beta-elemene	Breast cancer	MCF-7/DOX	↓ P‑gp	[81]
	Breast cancer	MCF-7/Adr and MCF-7/Doc cells	↓ P‑gp	[82]
Artesunate	Leukemia	CEM/ADR5000 and CEM/VCR100 cells	↓ P‑gp	[87]
	Lung cancer	A549 cells	↓ BCRP	[88]
	Esophageal cancer	Eca109/ABCG2 cells	↓ BCRP	[89]
Parthenolide	Lung cancer	A549/DOX cells	↓ P‑gp	[91]
	Hepatocellular carcinoma	BEL-7402/5-FU cells	↓ P‑gp, MRP	[92]
	Leukemia	K562/ADM cells and K562 cells	↓ P‑gp via NF-κB signaling	[93]
	Breast cancer	MDA-MB231 cells	↓ P‑gp	[94]
	Pancreatic cancer	GEM-resistant AsPC-1 and MIA PaCa-2 cells	↓ MRP1	[95]
Wilforine	Cervical cancer	HeLaS3 and KBvin cells	↓ P-gp	[97]
Tenulin and isotenulin	Cervical cancer	HeLaS3 and KBvin cells	↓ P-gp	[98]

Efficacy of diterpenoids as re-sensitizing agent

Numerous studies have identified terpenoids and aromatic chemicals in essential oils as important anticancer agents [99]. Diterpenoids, a subclass of terpenoids found in essential oils, are currently receiving attention for their potential biological activity [100]. Several prospective anticancer diterpenoids and related compounds have been recognized, including taxanes, triptolide, andrographolide, oridonin, and coffee derived diterpenoids [101–104]. The anticancer properties of these diterpenoids and their derivatives have been demonstrated in a number of studies, including in mice, humans, and various cell lines.

Triptolide, a diterpenoid triepoxide, is the principal active constituent of extracts from the plant Tripterygium wilfordii Hook F and is widely recognized for its significant anti-inflammatory, antitumor, and immunosuppressive effects [105]. Triptolide has been shown in numerous studies to suppress a number of human solid tumors both in vivo and in vitro [106]; it has also been shown to have a variety of anticancer mechanisms, including the inhibition of heat shock factor-1 (HSF-1), repression of the DNA damage response, and the regulation of mRNA stability [107–109]. A study showed that triptolide exhibits an antitumor impact on cancer KB cells and drug resistant KB cells, which overexpress MDR related proteins. The results indicated that triptolide reduces the expression of MDR proteins in both drug-resistant cell lines. It also triggers apoptosis in these cancer cells by stimulating caspase-3 and reducing Mcl-1 and XIAP levels. Triptolide not only suppresses tumor proliferation but also triggers apoptosis in xenograft mouse models [110]. There is evidence that triptolide administration inhibited MDR in prostate cancer cells by decreasing MDR1 expression. The findings indicated that triptolide suppressed the cell proliferation, induced apoptosis and halted cell cycle progression in DU145 cells. Triptolide reduced the levels of Cyclin D1 and Bcl-2 (anti-apoptotic protein) while elevating the expression of Fas and Bax (pro-apoptotic proteins). Moreover, triptolide restored the sensitivity of DU145/ADM cells to anticancer drugs, which was mediated by the suppression of MDR1 expression at both gene and protein levels. Altogether, these findings suggested that triptolide reverses MDR in prostate cancer cells by suppressing the expression of MDR1 [111]. A separate study indicated the impact of triptolide on adriamycin-resistant K562/A02 cells through modification of P-glycoprotein levels. T triptolide enhanced the aggregation of adriamycin in K562/A02 cells, substantially decreased P-gp expression and drug efflux function, and reduced the degree of resistance in these resistant cells. A luciferase reporter gene assay indicated that triptolide repressed the transcription function of MDR1 promoter. Triptolide may efficiently restore adriamycin resistance in drug resistant leukemia cells by downregulating P-gp level and enhancing intracellular adriamycin build-up [112].

Oridonin, a diterpenoid lactone obtained from medicinal plants, has attracted a lot of interest because of its medical potential, especially for its anticancer effects. It exhibits antitumor potential by triggering apoptosis, inhibiting cancer cell growth, and enhancing cellular sensitivity to standard chemotherapeutics by altering critical signaling pathways, such as PI3K/AKT, NF-κB, and MAPKs [113]. Research has revealed that the combination of oridonin and cisplatin (DDP) could reverse the chemoresistance of SGC7901/DDP cells. Oridonin markedly inhibited the proliferation and colony-forming ability of DDP-resistant human SGC7901/DDP cells, leading to enhanced caspase-mediated apoptosis and the downregulation of P-gp, MRP1, and cyclin D1 [114].

Carnosic acid is a phenolic diterpenoid commonly obtained from Lamiaceae plants, such as Rosmarinus officinalis L [115]. Recent research suggested that carnosic acid possesses remarkable anticancer properties against several tumors, including leukemia, colorectal, liver, pancreatic, stomach, breast, lung, prostate, and oral cancer. Carnosic acid could be associated with a number of potential antitumor pathways, such as the suppression of cell proliferation and metastasis, the apoptotic induction and autophagy, the regulation of the immune system and gut microbiota, and the enhancement of the sensitivity of chemodrugs [116, 117]. A study evaluated the influence of carnosic acid on multidrug-resistant (MDR)-associated antitumor mechanisms in leukemia CEM/ADR5000 and CCRF-CEM cells. Carnosic acid overcomes the resistance conferred by the expression of ABC transporters like P-gp, BCRP, and ABCB5 [118]. In 2010, a study examined the potential of rosemary phytocompounds, including carnosic acid, carnosol, rosmarinic acid, and ursolic acid, on the functionality of the drug efflux transporters. In this study, carnosic acid, carnosol, and ursolic acid were effective in stimulating the ATPase activity of P-glycoprotein. KB-C2 cells exhibited increased sensitivity to vinblastine cytotoxicity due to carnosic acid, highlighting that carnosic acid overcome MDR. These findings indicate that rosemary phytocompounds, including carnosic acid, block the drug efflux transporter P-gp and improve sensitivity to chemotherapy [119].

A diterpenoid lactone, andrographolide is the primary bioactive component of the Andrographis paniculata (Burm. f.) Nees plant, possess significant anticancer effects. Prior research has indicated the possible involvement of andrographolide in the modulation of oxidative stress, apoptosis, autophagy, suppression of cell proliferation, and migration [120, 121]. The chemosensitizing effect of andrographolide on P-glycoprotein-overexpressing multidrug-resistant KBChR 8 − 5 cells was assessed. A research has demonstrated that andrographolide displays superior binding to P-gp than the other two ABC transporters. Moreover, it suppressed P-gp transport in a concentration-dependent manner in KBChR 8 − 5 cells. Furthermore, andrographolide attenuated P-gp overexpression through NF-κB activation in these multidrug-resistant cell lines. The combination of andrographolide and paclitaxel demonstrated increased apoptotic cell death in KBChR 8 − 5 cells in comparison with paclitaxel alone [122]. Similarly, further study investigated the sensitizing effect of andrographolide in combating multidrug resistance in drug-resistant KBChR 8 − 5 cells. Andrographolide demonstrated enhanced cytotoxicity in P-gp-overexpressing KBChR 8 − 5 cells. Moreover, andrographolide exhibited synergistic effects with PTX and DOX in these drug-resistant cells. The combined administration of andrographolide augmented the cytotoxic effects of PTX and DOX while decreasing cell growth in multidrug-resistant cancer cells. Furthermore, ROS increased along with a reduction in mitochondrial membrane potential (MMP) following combination treatment with andrographolide and chemotherapeutic agents in drug-resistant cells. Additionally, andrographolide (4 µM) decreased the expression levels of ABCB1 and AKT [123].

A jatrophane diterpenoid, euphomelliferine, isolated from Euphorbia mellifera, was assessed for its ability to reverse multidrug resistance (MDR) associated with P-glycoprotein. This was conducted using a rhodamine-123 exclusion assay on mouse lymphoma cells and human colon adenocarcinoma cells. The phytochemical exhibited a substantial reversal of multidrug resistance through decreased P-gp expression across both cancer cell types [124]. Similarly, another jatrophane diterpenoid, euphodendroidin D, was assessed for its efficacy to reverse MDR associated with P-glycoprotein in leukemia K562/R7 cells. Euphodendroidin D exhibited considerable multidrug resistance reversal efficacy through P-glycoprotein downregulation in leukemia cells [125]. Moreover, the jatrophane diterpenoid pepluanin A can reverse MDR in leukemia cells by downregulating P-glycoprotein [126].

Tanshinones are a class of abietane diterpenes extracted from the traditional Chinese medicinal plant Salvia miltiorrhiza. Various tanshinones, such as dihydrotanshinone, cryptotanshinone, and miltirone, have demonstrated antitumor potential against multiple malignancies, including stomach, colon, and breast cancer [127, 128]. In addition, it has been demonstrated that cryptotanshinone and dihydrotanshinone are capable of overcoming drug resistance in colon cancer cells by reducing the expression of P-gp and promoting autophagic cell death [129–132].

Tanshinone IIA, a natural diterpene quinone derived from Salviae miltiorrhiza, has several biological activities. Tanshinone IIA exhibits extensive anticancer effects across several human tumor cell lines by decreasing tumor proliferation, inducing apoptosis, modulating the cell cycle, signaling transduction pathways, and overcoming MDR in different human cancer cells [133]. A study reported the ability of tanshinone IIA to circumvent stomach cancer cell resistance to the widely used anticancer drug doxorubicin. The results indicated that tanshinone II enhanced the anticancer efficacy of doxorubicin in gastric carcinoma cells by inhibiting MRP1 function and promoting cell cycle arrest, autophagy and apoptotic cell death [134]. Li and Lai demonstrated how Tan IIA could dose-dependently increase DOX’s antitumor impact on MCF-7 and MCF-7/DOX cells, particularly on MCF-7/DOX cells. Tan IIA can enhance intracellular Dox aggregation in breast cancer cells by decreasing P-gp, BCRP, and MRP1 levels, effectively eliminating cancerous cells, and thereby improving the susceptibility of breast cancer to chemotherapy [135]. Su demonstrated that combined treatment with Tanshinone IIA and 5-fluorouracil resulted in diminished xenograft tumor sizes and reduced expression of P-gp compared to 5-FU alone in a Colo205 xenograft model [136]. Research has exhibited that Tan IIA might augment the susceptibility of breast cancer cells to doxorubicin by regulating the PTEN/AKT pathway and decreasing the levels of P-gp, BCRP, and MRP1. Additionally, in vivo investigations demonstrated that Tan IIA augmented the chemotherapeutic efficacy of Dox in breast cancer while mitigating its adverse effects, such as weight loss, cardiotoxicity, and nephrotoxicity [137]. Furthermore, a study reported the effects of tanshinone ⅡA on the chemosensitivity of DOX against breast cancer cell lines MCF-7 and MDA-MB-231. In comparison to DOX alone, breast cancer cells administered with tanshinone ⅡA in conjunction with DOX displayed a reduced growth rate. Tanshinone IIA significantly augmented the capacity of DOX to trigger apoptotic cell death in breast cancer cells and eliminate breast cancer stem cells. This compound also facilitated the aggregation of DOX and decreased the expression of P-gp, BCRP, and MRP1 in breast cancer cells [138]. We have summarized the anticancer mode of action of diterpenoids in several carcinomas via downregulating P-gp, MRP1 and BCRP in Table 2.

Table 2.

Diterpenoids as resensitizing agent via P-gp, MRP1 and BCRP modulation

Diterpenoid	Cancer	Model	Target of drug resistance	References
Triptolide	Oral cancer	KB cells	↓ MRP and P-gp expressions	[110]
	Prostate cancer	DU145/ADM cells	↓ MDR1	[111]
	Leukemia	K562/A02 cells	↓ P-gp	[112]
Oridonin	Gastric cancer	SGC7901/DDP cells	↓ P‑gp, MRP1	[114]
Carnosic acid	Leukemia	CEM/ADR5000, CCRF-CEM cells	↓ P‑gp, BCRP	[118]
	Cervical cancer	KB-C2 cells	↓ P‑gp,	[119]
Andrographolide	Cervical cancer	KBChR 8 − 5 cells	↓ P‑gp	[122]
	Cervical cancer	KBChR 8 − 5 cells	↓ P‑gp	[123]
Euphomelliferine	Lymphoma and colon cancer	L5178Y mouse T-lymphoma cells, COLO 205 and COLO 320 MDR cells	↓ P‑gp	[124]
Euphodendroidin D	Leukemia	K562/R7 cells	↓ P‑gp	[125]
Pepluanin A	Leukemia	K562/R7 cells	↓ P‑gp	[126]
Cryptotanshinone and dihydrotanshinone	Colon cancer	Caco-2, SW620 SW620 Ad300 cells	↓ P‑gp	[131]
Tanshinone IIA	Gastric cancer	SNU-719R and SNU-601R cells	Inhibition of MRP1 function	[134]
	Breast cancer	MCF-7 cells and MCF-7/DOX cells	↓ P-gp, BCRP and MRP1	[135]
	Colon cancer	Colo205 cells; xenografted mice with Colo205 cells	↓ P-gp	[136]
	Breast cancer	MCF-7 and MCF-7/DOX cells; MCF-7 cells xenograft mouse model	↓ P-gp, BCRP, and MRP1	[137]
	Breast cancer	MCF-7 and MDA-MB-231 cells	↓ P-gp, BCRP, and MRP1	[138]

Efficacy of triterpenoids as re-sensitizing agent

Triterpenoids are organic molecules classified according to their structural diversity. They are distinguished by a fundamental backbone that can be altered in a number of ways, which enables the development of more than 20,000 known members. Triterpenoids are produced from isopentenyl pyrophosphate via the 30-carbon transition product, squalene. Triterpenoids have garnered significant interest as potential natural anticancer compounds. Several studies have documented the anticancer and apoptosis-inducing properties of triterpenoids. Triterpenoids exhibit significant multitarget ability, and their antitumor efficacy is exhibited by their capacity to prevent NF-κB activation, repress cellular proliferation, angiogenesis, signal transduction and regulate MDR associated targets [139, 140]. Amyrin terpenes have been documented to have considerable anticancer and cytotoxic effects [141, 142]. 3-oxo-6β-hydroxy-β-amyrin is a natural triterpenoid of the amyrin class, and is known for its substantial β-secretase, α-glucosidase, and anticancer activities [143]. A study investigated the efficacy of 3-oxo-6β-hydroxy-β-amyrin in reversing P-gp-mediated multidrug resistance in Raji leukemia cells. The findings indicated that 3-oxo-6β-hydroxy-β-amyrin functioned as an efficient MDR modulator through downregulating P-gp in leukemia cells [144].

In 2008, a study reported the impact of dietary phytocompounds on the activity of P-gp and MRP1 to combat multidrug resistance by employing ABC transporters expressing leukemia cells. In this study, glycyrrhetinic acid enhanced the aggregation of calcein (MRP1 substrate) in KB/MRP cells. These two cell lines (KB-C2 and KB/MRP) were sensitized to chemotherapy drugs by glycyrrhetinic acid, demonstrating that glycyrrhetinic acid overcomes MDR. These findings indicate that dietary phytochemicals, including glycyrrhetinic acid from licorice, exhibit parallel suppressive effects on P-gp and MRP1, potentially enhancing the efficiency of cancer treatment [145]. Research has demonstrated potent P-gp-mediated MDR suppression activity of obacunone in MES-SA/DX5 and HCT15 cells, with ED50 values of 0.028 and 0.0011 µg/mL, respectively [146].

The anticancer efficacy of three pentacyclic triterpenoids, betulinic, oleanolic, and pomolic acids, derived from Chrysobalanaceae species, has been documented in both multidrug-resistant and sensitive leukemia cell lines. These results indicated that these triterpenoids were more effective in suppressing P-gp expression in leukemia cells [147]. Triterpenoids uvaol and oleanolic acid extracted from the methanolic extract of Carpobrotus edulis have been shown to inhibit P-gp in MDR1-transfected murine lymphoma cells [148].

There is evidence that alisol B 23-acetate recovered the susceptibility of multidrug-resistant cell lines HepG2-DR and K562-DR to antineoplastic drugs with diverse mechanisms of action, all of which are substrates of P-glycoprotein. This triterpenoid recovered the efficacy of vinblastine, a P-glycoprotein substrate, in inducing G2/M arrest in multidrug-resistant cells. Alisol B 23-acetate enhanced DOX retention and inhibited the efflux of rhodamin-123 from MDR cells. Alisol B 23-acetate reduced the photoaffinity labeling of P-glycoprotein and enhanced the ATPase activity of P-glycoprotein, indicating that it may serve as a substrate for P-glycoprotein transport. Moreover, when verapamil was employed as a substrate, alisol B 23-acetate exhibited partial noncompetitive inhibition of P-gp [149].

20(S)-ginsenoside Rg3 (Rg3) is a ginsenoside recognized for its inhibitory effects on several tumors, including those of the lung, gallbladder, liver, and ovary [150]. Moreover, it has been documented that Rg3 modulates multidrug resistance in many tumor cells, particularly human acute myeloid leukemia, KBV20C, and murine leukemia P388 cells [151, 152]. Research has shown that Rg3 administration suppressed the levels of MDR-related proteins, namely P-gp, MRP1, and lung resistance protein 1 (LRP1) in DDP-resistant A549 cells (A549/DDP). Moreover, Rg3 enhances the anticancer efficacy of DDP in A549/DDP xenograft mice [153].

Amooranin, another tripterpenoid derived from Amoora rohituka, has been shown to reverse drug resistance through P-gp suppression in colon cancer and leukemia cells. Co-treatment of cancer cells with both DOX and amooranin restored drug resistance in these cancer cell lines. Expression level of P-gp was inhibited by amooranin, and this blocking effect is amplified as the quantity of amooranin increases [154].

Additionally, the reversal effect of the triterpenoid ursolic acid on multidrug resistance and the associated processes were examined in drug-resistant breast and ovarian cancer cells. A study reported that ursolic acid could enhance the intracellular accumulation of DOX in the nucleus and reduce the efflux ratio of digoxin, similar to the effects of the established inhibitor verapamil, by functioning as a P-gp substrate in MDR MCF-7/ADR cells [155]. Furthermore, a study indicated that ursolic acid suppressed cellular proliferation and reverses MDR in ovarian CSCs by declining HIF-1α and ABCG2 expression [156].

A triterpenoid from the citrus family, limonin, has been studied for its ability to alter P-gp activity in Caco-2 colon cancer cells and the MDR human leukemia cell line CEM/ADR5000. Administration of limonin triterpenoid to drug-resistant Caco-2 and CEM/ADR5000 cells enhanced their susceptibility to DOX and completely recovered DOX sensitivity, correlating with reduced P-glycoprotein activity [157]. Additionally, the anticancer efficacy of the neem triterpenoid nimbolide was assessed by reversal of multidrug resistance in MDR1-expressing CEM/ADR5000 leukemia cells. The CEM/ADR5000 cell line, which overexpresses P-glycoprotein (ABCB1/MDR1), exhibited noteworthy hypersensitivity to nimbolide, mediated by the enhanced expression of the tumor suppressor PTEN and its downstream components, ultimately leading to a substantial reduction in ABCB1/MDR1 mRNA and P-gp [158]. We have summarized the anticancer mode of action of triterpenoids in several carcinomas via downregulating P-gp, MRP1 and BCRP in Table 3.

Table 3.

Triterpenoids as resensitizing agent via P-gp, MRP1 and BCRP modulation

Triterpenoid	Cancer	Model	Target of drug resistance	Reference
3-oxo-6β-hydroxy-β-amyrin	Lymphoma	Raji cells	↓ P-gp	[144]
Glycyrrhetinic acid	Cervical cancer	KB-C2 cells	↓ P-gp and MRP1	[145]
Obacunone	Lung, ovarian and colon cancer, melanoma	A549, SK-OV-3, SK-MEL-2, and HCT15 cells	↓ P-gp	[146]
Oleanolic acid	Leukemia	K562 cells	↓ P-gp	[147]
	Lymphoma	L5178	↓ P-gp	[148]
Betulinic acid	Leukemia	K562 cells	↓ P-gp	[147]
Pomolic acid	Leukemia	K562 cells	↓ P-gp	[147]
Uvaol	Lymphoma	L5178	↓ P-gp	[148]
β-amyrin	Lymphoma	L5178	↓ P-gp	[148]
Alisol B 23-acetate	Hepatocellular carcinoma and leukemia	HepG2 and K562 cells	↓ P-gp	[149]
Ginsenoside Rg3	Lung cancer	A549/DDP cells; A549/DDP-xenograft mice	↓ P-gp, MPR1 and LPR1	[153]
Amooranin	Leukemia and colon cancer	CEM/VLB and SW620/Ad-300 cells	↓ P-gp	[154]
Ursolic acid	Breast cancer	MCF-7/ADR cells	Inhibition of P-gp function	[155]
	Ovarian cancer	SKOV3, A2780, and HEY cells	↓ BCRP	[156]
Limonin	Colon cancer and Leukemia	Caco2 and CEM/ADR5000 cells	↓ P‑gp	[157]
Nimbolide	Leukemia	CEM/ADR5000 cells	↓ P-gp, and MRP1	[158]

Conclusion and future perspectives

The predominant mechanism of multidrug resistance (MDR) is the active efflux of chemodrugs by ATP-binding cassette (ABC) transporters. P-gp, MRP1, and BCRP impart resistance to several chemotherapeutic agents. Recent investigations into drug resistance-associated proteins have yielded encouraging results in overcoming MDR by emphasizing cell membrane transport proteins. Significant efforts have been made to identify and generate inhibitors that can target these proteins. Consequently, effective and safe inhibitors must be discovered for therapeutic applications in the reversal of MDR. Terpenoids, which are mostly present in many plants, have the advantages of non-toxicity and multitarget approaches. A growing body of research indicates that terpenoids may act as sensitizing agents when administered alone or in combination. The primary challenge in studying the mechanisms of terpenoids is the insufficient understanding of their specific targets and modes of action in overcoming resistant cancer cells. Existing research, such as that reviewed above, indicates that these terpenoids exhibit antitumor activity via several mechanisms and multiple targets. It is quite apparent that certain terpenoids may be able to combat MDR by modulating numerous factors that promote MDR. Terpenoids may modulate ABC transporters including ABCB1, ABCG2, and ABCC1. In conclusion, terpenoids have demonstrated potential as natural, cost-effective, and non-toxic agents capable of reversing MDR by blocking ABC efflux pumps in several carcinomas.

However, several problems have emerged in the real-world implementation of plant-derived terpenoids that have mediated the reversal of MDR. Although the advancement of MDR reversal drugs has progressed significantly, it is crucial to acknowledge that P-gp and other associated transport proteins are present not only in cancerous cells, but also extensively localized in normal cells. Therefore, achieving equilibrium between obstructing the efflux of anticancer agents and preserving homeostasis is essential for efficient application of MDR reversal drugs. Another issue is the instability and bioavailability of drug-delivery systems that carry sensitizing drugs. Consequently, additional research is required to ascertain methods for maintaining the efficacy of the drug within effective drug carriers and facilitating their successful release at the target site. Additionally, it is crucial to emphasize that most current research on this topic is in the in vitro experimental phase. Consequently, comprehensive in vivo research and clinical trials are essential for confirming the efficacy and reproducibility of these results. Given the multitarget characteristics of plant-derived terpenoids, it is crucial to further investigate the mechanisms of action of the novel semisynthetic derivatives and to uncover additional targets beyond ABC proteins that may also play a role in reversing complex MDR phenomena.

Author contributions

P.P., A.S, S.L., M.V., and F.K. conceived and designed this study. R.M., V.J.U., S.M., and F.K. supervised this study. P.P., A.S., and F.K. prepared the manuscript. R.M., S.M., S.L., and V.J.U. revised the manuscript. All authors read and approved the final manuscript.

Funding

None.

Data availability

No datasets were generated or analysed during the current study.

Declarations

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Not applicable.

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Competing interests

The authors declare no competing interests.