Chemosensitizing potential of andrographolide in P-glycoprotein overexpressing multidrug-resistant cancer cell lines

Abstract The P-glycoprotein (P-gp) plays a major role in the efflux of chemotherapeutic drugs and significantly limits chemotherapy efficacy. Chemosensitizers augment the therapeutic effects of anticancer agents by overcoming drug resistance mechanisms. In this study, the chemosensitizing property of andrographolide (Andro) in P-gp overexpressing multidrug-resistant (MDR) colchicine-selected KBChR 8-5 cells was evaluated. Molecular docking studies showed Andro exhibits higher binding interaction with P-gp than the other two ABC-transporters studied. Further, it inhibits P-gp transport function in a concentration dependant manner in the colchicine-selected KBChR 8-5 cells. Moreover, Andro downregulates P-gp overexpression via NF-κB signaling in these MDR cell lines. MTT-based cell-based assay illustrates that Andro treatment augments the PTX effect in the KBChR 8-5 cells. Further, the Andro plus PTX combination showed enhanced apoptotic cell death in KBChR 8-5 cells compared with PTX alone treatment. Therefore, the results showed that Andro enhances PTX therapeutic effect in the drug-resistant KBChR 8-5 cells. Graphical Abstract


Introduction
Multidrug resistance (MDR) has become a severe impediment in cancer chemotherapy.Overexpression of ATP binding cassette (ABC) transporters in the cancer cells resulted in the development of clinical MDR (Braconi et al. 2022).The P-glycoprotein (P-gp) is the well-studied drug efflux ABC transporter that contributes to the development of MDR in cancer cells.P-gp effluxes out a wide range of drugs including alkaloids such as vinblastine, anthracyclines, epipodophyllotoxins, etc. (Cardoso et al. 2021).The P-gp inhibitors bind with the drug-binding pocket of the transmembrane domain thereby inhibiting its drug efflux transport function.Inhibition of P-gp transport function or downregulation of its overexpression seems to be a novel approach to overcome the clinical MDR in cancer cells (Amin 2013).Several generations of small molecule inhibitors have been invented for the reversal of P-gp-mediated MDR (Prasad et al. 2016).However, the currently available P-gp inhibitors inadvertently accumulate chemotherapeutic drugs in the non-targeted normal cells.Hence, researchers are involved in the development of P-gp inhibitors/modulators with improved specificity to P-gp in cancer cells and their non-toxic nature in normal cells (Marques et al. 2021).Recent reports illustrate that phytochemicals effectively sensitize the MDR cancer cells to different chemotherapeutic agents (Bijani et al. 2022).
Andrographolide (Andro) is a labdane diterpenoid, aqueous insoluble phytochemical present in the Andrographis paniculata 'the king of bitterness' (Lu et al. 2019).Andro, exhibit several pharmacological properties in multidrug resistant cells like oxidative cellular damage and anti-inflammatory action (Tan et al. 2018;Burgos et al. 2020).It modulates mitogen-activated protein kinase (MAPK) signaling, Nuclear factor-κB (NF-κB), and signal transducer and activator of transcription 2 (STAT3) activation in the experimental models (Li et al. 2022).The NF-κB plays an important role in promoting drug resistance in cancer cells.The expression of NF-κB in cancer cells indicates resistance to apoptotic induction by chemotherapeutic drugs (Abdin et al. 2021).Studies indicate that NF-κB signaling contributes for the overexpression of ABC transporters in the MDR cancer cells (Ganesan et al. 2021).Andro has been reported to improve the radiosensitivity and chemosensitivity in different cancer cells (Li et al. 2020;Sharma et al. 2020).In this study, we investigated the chemosensitizing effect of Andro in P-gp-overexpressing KBCh R 8-5 cells via targeting NF-κB nuclear translocation.

Andro binding interactions with P-gp, BCRP, MRP1, and ABCB4
We observed that Andro had a greater binding affinity with human P-gp (-6.0 kcal/ mol) compared to other ABC transporters MRP1 and BCRP (Supplementary material Table S1).The human P-gp has a larger binding pocket (6000 Å) with primarily aromatic and hydrophobic residues, therefore, exhibits higher binding affinity to the ligands (Nobili et al. 2020).Andro interacts with the drug-binding domain of P-gp through both hydrogen bonding and hydrophobic interactions ALA 995 and SER 993 (Supplementary material Figure S1(a)).Another crucial interaction was the alkyl bond interaction at the PRO 996 position (Supplementary material Figure S1(a)).

Andro inhibits P-gp efflux in KBCh R 8-5 cells
We tested the inhibitory potential of Andro against P-gp-mediated drug efflux function.We observed that Andro reduced the P-gp transport activity in a concentration dependant manner (2 µM − 10 µM) in P-gp overexpressing KBCh R 8-5 cells (Supplementary material Figure S2(a)).Spectrofluorometric data further confirm the inhibition of P-gp transport activity by Andro in a concentration dependant manner, and it was found to be highest at 10 µM Andro treatments (87 ± 5.4%) (Supplementary material Figure S2(b)).Similarly, bioactive phytochemicals like ferulic acid and celastrol inhibit P-gp transport function and accumulate the fluorescent substrate calcein-AM in KBCh R 8-5 cells (Muthusamy et al. 2016).

Andro downregulates ABCB1and NF-κB p50 expression in resistant KBCh R 8-5 cells
In this study, the colchicine-selected KBCh R 8-5 cells prominently showed the membrane overexpression of P-gp (Supplementary material Figure S3).Phytochemicals modulate cell signaling pathways thereby reversing P-gp-mediated MDR.The immunocytochemistry studies revealed that Andro significantly downregulated the overexpression of P-gp probably by inhibiting the translocation of the NF-κB p50 subunit from the cytoplasm to the nucleus (Supplementary material Figure S4).Phytochemicals inhibit the translocation of NF-κB, thereby reversing P-gp-mediated MDR in tumor cells (Muthusamy et al. 2019).Moreover, Andro was earlier stated as an inhibitor of the NF-κB signaling pathway and reduced transcriptional activity (Soleimani et al. 2020;Burgos et al. 2020).

Chemosensitization by Andro in KBCh R 8-5 cells
The chemosensitivity of Andro in combination with PTX was evaluated in the parental KB 3-1 and KBCh R 8-5 cells (Supplementary material Figure S5(a,b)).Andro augments the sensitivity of KB 3-1 and KBCh R 8-5 cells to the chemotherapeutic drug in a concentration-dependent manner.The P-gp overexpressing KBCh R 8-5 cells showed minimal sensitivity to PTX with an IC 50 value of 1.628 ± 1.296 µM, and the fold resistance (FR) was calculated as 3.57 (Supplementary material Table S2).The PTX fold resistance (FR 3.57) was decreased to 1.95, 0.69, and 0.30 during 1, 2, and 4 µM of Andro treatment, respectively.Earlier a study states that Andro along with carboplatin act synergistically in Hep-2 human laryngeal carcinoma cells and showed higher cell growth retardation than carboplatin alone (Mao et al. 2019).

Andro-induced cell death in KB 3-1 and KBCh R 8-5 cells
In this study, a higher rate of apoptosis was observed in the Andro and PTX combination than in the untreated group revealing the chemosensitivity activity of Andro in resistant KBCh R 8-5 cells (Supplementary material Figure S6(a)).Apoptotic cells were higher in the parental KB 3-1 cell (74.9%) (Supplementary material Figure S6(b)).The resistant KBCh R 8-5 cells had 50.5% of apoptotic cells in the combinational treatment group with Andro (4 M) and PTX (0.1 M) (Supplementary material Figure S6(b)).A previous study suggested that Andro potentiates the cisplatin activity that enhances apoptosis in the A549 NSCLC cells (Hong et al. 2022).Treatment with Andro raised apoptotic cells in MDA-MB-231 and MCF-7 cells (Banerjee et al. 2016).Moreover, Andro sensitizes HCT116 colorectal cancer cells by regulating P13K-Akt-mTOR signaling to radiotherapy (Li et al. 2020).

Andro affects cell migration in KB 3-1 and KBCh R 8-5 cells
Furthermore, the Andro and PTX combination inhibited the cell migration and proliferation of sensitive KB 3-1 and resistant KBCh R 8-5 cells (Supplementary material Figure S7(a-d)).The difference in the migratory behaviour of cells in the control group and the Andro and PTX combination indicates the chemosensitizing potential of Andro.Liao et al. in their study on human oral epidermoid carcinoma (OEC-M1) cells stated that the migratory rate is related to the inhibitory effect of Andro in a concentration-dependent manner (Liao et al. 2022).Andro remarkedly showed less cell migration in the dose-dependent manner in SGC7901, 5637 and T24 cells by interfering with the NF-κB signaling pathway (Dai et al. 2017;Xuan et al. 2022).Thus, Andro exhibits a chemosensitizing effect in the colchicine-selected P-gp overexpression experimental cell line.

Conclusion
The present results demonstrate that Andro downregulates the expression of P-gp in resistant KBCh R 8-5 cells.This downregulation of P-gp is probably through the inhibition of NF-κB translocation to the nucleus.Further, Andro enhances therapeutic efficacy of PTX in the MDR cells.Therefore, Andro can be used as a potential P-gp inhibitor to overcome MDR in cancer cells.Further studies are required to clarify the relationship between Andro and the signaling pathway involved in the P-gp modulation in tumor xenografts animal models.