Andrographis-mediated chemosensitization through activation of ferroptosis and suppression of β-catenin/Wnt-signaling pathways in colorectal cancer

Abstract

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality in the USA. As much as 50–60% of CRC patients develop resistance to 5-fluorouracil (5FU)-based chemotherapeutic regimens, attributing the increased overall morbidity and mortality. In view of the growing evidence that active principles in various naturally occurring botanicals can facilitate chemosensitization in cancer cells, herein, we undertook a comprehensive effort in interrogating the activity of one such botanical—andrographis—by analyzing its activity in CRC cell lines [both sensitive and 5FU resistant (5FUR)], a xenograft animal model and patient-derived tumor organoids. We observed that combined treatment with andrographis was synergistic and resulted in a significant and dose-dependent increase in the efficacy of 5FU in HCT116 and SW480 5FUR cells (P < 0.05), reduced clonogenic formation (P < 0.01) and increased rates of caspase-9-mediated apoptosis (P < 0.05). The genomewide expression analysis in cell lines led us to uncover that activation of ferroptosis and suppression of β-catenin/Wnt-signaling pathways were the key mediators for the anti-cancer and chemosensitizing effects of andrographis. Subsequently, we validated our findings in a xenograft animal model, as well as two independent CRC patient-derived organoids—which confirmed that combined treatment with andrographis was significantly more effective than 5FU and andrographis alone and that these effects were in part orchestrated through dysregulated expression of key genes (including HMOX1, GCLC, GCLM and TCF7L2) within the ferroptosis and Wnt-signaling pathways. Collectively, our data highlight that andrographis might offer a safe and inexpensive adjunctive therapeutic option in the management of CRC patients.

Introduction

Colorectal cancer (CRC) is the third most common cancer in the USA, with >147 950 new cases diagnosed and ~53 200 deaths each year, making it the second leading cause of cancer-related deaths among adults (1). Although 5-fluorouracil (5FU)-based chemotherapy remains the mainstay for treating majority of CRC patients (2), intrinsic and acquired resistance to this treatment modality presents with a major clinical challenge (3). More importantly, the response rates to 5FU-based monotherapy and combination therapies in advanced CRC patients vary between a dismal 10–15% and 40–50%, respectively (4). Chemotherapeutic drug resistance often stems from the small pool of mutant cells that acquire selection and growth advantage during the treatment course (5). In view of this limitation, combination therapies offer a distinct advantage as they eliminate mutant cell fractions often resistant to monotherapies and decrease the risk for the emergence of clones that are resistant to multiple drugs (6). Mathematical modeling analysis has supported this concept and has shown that the efficacy of drugs can be improved from 0 to 88% through the use of a single versus combinatorial therapies in various cancers (7). In addition to the minimal response rates in CRC, 5FU-based treatments are frequently associated with severe side effects including hematological suppression, as well as gastrointestinal, hepatic and renal damage (8,9), highlighting the need for the development of safer and more effective treatment modalities in CRC patients.

The use of natural product-based, complementary and alternative treatment approaches have become popular in cancer medicine during the past two decades, primarily due to their inherent safety profile (10,11). In addition, natural product-derived anti-cancer compounds exert their biological activity in a multitargeted manner, thereby influencing multiple growth regulatory pathways that inhibit cancer cell growth on their own, or work as adjuncts along with conventional chemotherapies in improving their overall therapeutic efficacy (12). In this context, andrographolide, a bicyclic diterpenoid lactone, is an active principle component of the medicinal herb, Andrographis paniculata, which is a member of the Acanthaceae family. Andrographis has been reported to possess wide-ranging biological activities including immunomodulatory (13), anti-inflammatory (14,15), anti-oxidant (16) and anti-cancer effects (17,18). In addition to its anticancer activity by itself, recent evidence also indicates that andrographolide treatment can overcome de novo chemoresistance and resensitize cancer cells to various chemotherapeutic drugs (19–21). For instance, andrographolide-based adjunctive treatment, together with conventional cytotoxic drugs including 5FU, cisplatin and gemcitabine, improved the overall chemotherapeutic response in multiple cancers (22–24). In addition, when used in combination, andrographolide-based treatment led to significant reduction in the dosage of individual chemotherapeutic drugs, diminishing their side effects while enhancing the therapeutic efficacy of these agents in the cancer cells (20,25). However, the molecular mechanisms underpinning the anti-cancer effects of andrographis, as well as its ability to resensitize CRC cells to 5FU-based chemotherapy, remain largely unclear.

Accordingly, in the present study, using a series of systematic and comprehensive approach by analyzing cell lines, a xenograft animal model and patient-derived tumor organoids, we interrogated whether andrographis has any chemosensitizing potential in CRC and the molecular mechanisms responsible for its anti-cancer activity. Although some of the previous studies have interrogated specific molecular pathways regulated by andrographis (15–18,26), none of the studies to date have performed an unbiased, comprehensive, genomewide interrogation for its activity in CRC. To address this important gap in knowledge, we for the first time performed a genomewide expression profiling of naive and 5FU-treated CRC cells to identify the most relevant cell-signaling pathways responsible for mediating chemosensitizing effects of andrographis in this malignancy. Through a series of systematic interrogations, we provide a novel evidence that andrographis-mediated sensitization to 5FU-based chemotherapy in CRC is primarily mediated through activation of ferroptosis and suppression of β-catenin/Wnt-signaling pathways, highlighting its potential adjunctive therapeutic role in the management of patients suffering from this fatal malignancy.

Materials and methods

Cell lines and materials

CRC cell lines, HCT116 and SW480, were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were tested and authenticated using a panel of genetic and epigenetic markers and evaluated for mycoplasma on a regular basis. The cell lines were grown in Iscove’s modified Dulbecco’s media (Gibco, Carlsbad, CA), supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and maintained at 37°C in a humidified incubator at 5% CO₂.

5FU (Sigma–Aldrich, St. Louis, MO) and andrographis extract (standardized to 20% andrographolide content; Andrographis EP80 was generously provided by EuroPharma USA, Green Bay, WI) were dissolved in dimethyl sulfoxide and diluted to appropriate experimental concentrations in the cell culture medium before use. The 5FU-resistant clones from both HCT116 and SW480 cells (HCT116 5FUR and SW480 5FUR) were established by continuous culturing of cells with increasing dose of the chemotherapeutic drug for >9 months, as described previously (27,28).

Cell viability, clonogenic assays and apoptosis analysis

All cells were plated in 96-well plates at a density of 3000 cells/well in Iscove’s modified Dulbecco’s media supplemented, and the cells were allowed to adhere to the plate surface overnight. Cell proliferation was measured in cells treated with a combination of 5FU (10, 20, 30, 40 µM) and andrographis (10, 20, 30, 40 ng/µl) for 72 h using the WST-1 assay (Sigma–Aldrich). Each experiment was performed in triplicates. To assess synergism between 5FU and andrographis on cell viability, a combination index (CI) that achieved 50% growth inhibition was calculated using the Chou–Talalay’s equation. A CI index of <1 was considered to be synergistic.

For clonogenic assays, the cells were seeded into six-well plates (300 cells/well) and were treated with andrographis, 5FU or their combination for 24 h once cells were adherent to the plate. Following 8 days, the number of colonies with >50 cells were counted, and the relative change in clonogenic survival for different treatment groups was determined. All experiments were conducted in triplicate.

For apoptosis analysis, cells were plated in six-well dishes and treated with 5FU, andrographis and their combination, for 48 h, in triplicates. Apoptosis assays were performed using the Muse Annexin-V and Dead Cell Assay kit (MCH100105, Millipore, Chicago, IL) on a Muse Cell Analyzer (Millipore), as per manufacturer’s instructions.

Gene expression profiling using microarray and pathway enrichment analysis

RNA was isolated from 2 × 10⁶ parental and 5FUR HCT116 and SW480 cells using miRNeasy kit (Qiagen, Hilden, Germany). RNA extracts with RNA integrity number values > 9.5 were used for gene expression profiling studies, using the Clariom S human gene expression arrays. Probe set selection was performed using detected above background values of ≤0.05 in >50% of samples. The raw expression data were preprocessed using robust multiarray average method in Transcriptome Analysis Console (TAC 4.0.1). Differential expression analysis was performed to identify significantly (P ≤ 0.05) differentially expressed genes using ebayes, using a threshold of P < 0.05 and |log FC| > 1.

For the identified differentially expressed genes, we performed Gene Ontology and KEGG pathway enrichment analysis using the R package ‘clusterProfiler’, and gene sets with a P value of <0.05 were considered significantly enriched.

mRNA expression analysis using RT–qPCR

RNA extraction from the cell lines, CRC organoids and xenograft tumors, treated with dimethyl sulfoxide (vehicle), 5FU, andrographis and a combination of the two compounds was performed using the miRNeasy kit (Qiagen). Extracted RNA was used as a template for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA). The RT-qPCR was performed using the SensiFAST SYBR mix (Bioline, London, UK) using the primer sequences listed in Supplementary Table 1 (available at Carcinogenesis Online). The relative expression for target genes was calculated using ΔΔCt method normalized against the housekeeping β-actin gene.

Western blot analysis

For western blot analysis, cells were treated with andrographis and/or 5FU for 48 h and lysed using RIPA buffer supplemented with proteinase inhibitor cocktail and denatured with 2× Laemmli’s sample buffer (Bio-Rad Laboratories, Hercules, CA), which contained 5% 2-mercaptoehanol (Sigma–Aldrich). The list of primary antibodies used for various experiments is provided in Supplementary Table 2 (available at Carcinogenesis Online). An anti-mouse IgG-HRP (sc-2005; 1:5000; Santa Cruz Biotechnology, Dallas, TX) or anti-rabbit IgG-HRP (sc-2004; 1:5000; Santa Cruz Biotechnology) were used as secondary antibodies. All samples were compared against β-actin (A5441, Sigma–Aldrich) as a reference protein. Chemiluminescence images were obtained using ChemiDoc-MP Imaging system (ver 5.2.1, Bio-Rad Laboratories Inc, Hercules, CA), and the band intensities were quantified using the Image J software ver. 1.52 (NIH, Bethesda, MD). A ratio of band intensity of target protein versus β-actin was determined, and the fold change with respect to the naive, untreated cells was determined.

Xenograft animal model

Seven-week-old male athymic nude mice (Envigo, Houston, TX) were housed under controlled conditions of light and fed ad libitum. Approximately 5 × 10⁶ parental and 5FUR HCT116 cells were suspended in the matrigel matrix (BD Biosciences, Franklin Lakes, NJ) and subcutaneously injected into mice using a 27-gauge needle (n = 10 per group). Mice were randomly assigned to different treatment groups and 5FU (30 mg/kg body weight) or andrographis (125 mg/kg body weight) or their combination were given intraperitoneally on alternative days for up to 15 days. Tumor size was measured each day by calipers. Tumor volume was calculated using the following formula: 1/2(length × width × width). The investigator was not blinded to the group allocation during the experiment and/or when assessing the outcomes. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Baylor Scott & White Research Institute, Dallas, TX. All experiments were conducted strictly in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th Edition Institute for Laboratory Animal Research). The human equivalent dose was calculated using formula as described previously (29).

Patient-derived tumor organoids

Fresh tumor tissues were obtained from CRC patients enrolled at the Baylor University Medical Center, Dallas, TX. The study was approved by the Institutional Review Board of the institution. A written informed consent was obtained from all patients providing tissue specimens, and all experiments were performed in accordance with relevant guidelines and regulations proposed in the Declaration of Helsinki. The clinicopathological characteristics of the patients from whom the CRC tissues were obtained are listed in Supplementary Table 3 (available at Carcinogenesis Online). CRC tumor organoids were cultured using a modified protocol described previously (30). For treatments, appropriate concentration of 5FU, andrographis and their combination were added to the culture medium, and tumor organoids were allowed to grow for 1 week. The experiment was performed in triplicates. The organoids were observed under a bright-field microscope. All 3D cell structures that were about 500 µm in diameter were counted. Organoids in a certain plane of field were counted, while leaving out the ones that were out of focus.

Statistical analysis

All experiments were repeated three times. All data were expressed as mean ± SD. Paired t-tests were performed to compare control versus treatment groups, and the alpha value of 0.05 was used to indicate the significance.

Results

Andrographis treatment sensitizes chemoresistant CRC cell lines to 5FU

In order to assess the effect of andrographis and 5FU in CRC cells, we first performed cell viability assays in parental and chemoresistant HCT116 and SW480 cell lines. We treated these cell lines individually with andrographis (10–40 ng/µl) and 5FU (10–40 µM), as well as their combination to determine whether there was any synergism between the two compounds. Not surprisingly, we observed that 5FU exhibited reduced cell viability compared with andrographis in both parental cell lines; however, the combination of the two compounds moderately increased the cytotoxicity in both cell lines (Figure 1A). In contrast, as expected, andrographis exerted a significantly greater cytotoxicity in 5FUR cell lines compared with 5FU, whereas the combination treatment exhibited synergism and a significantly reduced cell viability in both cell lines (Figure 1B). Furthermore, we observed that 5FU treatment reduced cell viability of the parental HCT116 and SW480 cell lines in a dose-dependent manner with a corresponding IC₅₀ concentration of ~4 µM, whereas their respective 5FU-resistant counterpart cell lines did not reach IC₅₀ concentrations during this treatment duration. Interestingly, combination treatment with andrographis in 5FUR cell lines lead to a significant reduction in the IC₅₀ concentration for 5FU (~7.5 µM) in HCT116 5FUR and SW480 5FUR cells lines (P < 0.05; Figure 1B).

Figure 1.

Andrographis sensitizes 5FU-resistant (5FUR) CRC cells. MTT assay was performed to compare cell viability against 5FU, andrographis and combination in (A) parental and (B) 5FUR CRC cell lines. Chou–Talalay CI shows whether combination of andrographis and 5FU is synergistic. (C) Isobologram representing CI < 1 and synergism between 5FU and andrographis in 5FUR CRC cells. (D) Data from the colony formation assay to assess clonogenicity of parental and 5FUR CRC cells following the treatment with vehicle, 5-FU, andrographis and combination for 7 days. A significant decrease in colony formation potential of CRC cells was observed after treatment with combination of andrographis and 5FU when compared with only 5FU group in parental and 5FUR CRC cells. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Chou–Talalay CI analysis demonstrated a synergistic effect between andrographis and 5FU in both resistant cell lines (CI < 1), suggesting that andrographis sensitized both chemoresistant cell lines to the effects of 5FU treatment (Figure 1C). In addition, we calculated the Dose Reduction Index to measure the extent to which the dose of 5FU can be reduced when treated in combination with andrographis than when used individually. As illustrated in Supplementary Table 4 (available at Carcinogenesis Online), the Dose Reduction Index values in each instance were >1 in both HCT116 and SW480 5FUR cell lines, highlighting the significant benefit for adjunctive treatment with andrographis in reducing the toxicity with a concurrent anti-cancer activity in CRC cells.

Next, we evaluated the combinatorial effects of andrographis and 5FU on cell survival using a clonogenic assay. As expected, 5FU treatment dramatically reduced colonosphere formation in parental cell lines (Figures 1D), whereas 5FUR cell lines were more resistant to the effects of the chemotherapeutic drug. More importantly, the combination treatment with andrographis was significantly more effective in inhibiting the colony formation in both cell lines (P < 0.05–0.01; Figure 1D), highlighting the superiority of the anti-cancer efficacy of the combination treatment with andrographis and 5FU in CRC cells.

Andrographis induces apoptosis through caspase-9 activation in chemoresistant CRC cells

Next, we questioned whether the anti-cancer effects of andrographis are in part mediated through enhanced apoptosis in parental and 5FU-resistant cell lines. We performed Annexin-V binding assays in various cell lines and observed that treatment with andrographis and 5FU resulted in significantly increase apoptosis in both HCT116 and SW480 cell lines and the combination of the two agents further elevated the apoptotic cell population when compared with only 5FU (P < 0.001; Figure 2A and B).

Figure 2.

The combination of andrographis and 5FU has superior anti-cancer effects compared with individual compounds. Representative images showing percentage of cells undergoing apoptosis as measured by percentage that stained positive for annexin-V assay in (A) parental and (C) 5FUR CRC cells. Histogram showing percentage of live, early apoptotic, late apoptotic and dead cell population in (B) parental and (D) 5FUR CRC cells. (E) The figure illustrates mRNA levels of caspase-9 in parental and 5FUR CRC cells treated with andrographis and 5FU in combination or individually. mRNA levels of β-actin were used as the internal normalizing control, and fold change was calculated when compared with cells treated with dimethyl sulfoxide control. Statistical Significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Although treatment of cells with 5FU had a limited effect on apoptosis in 5FUR cell lines, the addition of andrographis in these cells significantly elevated apoptosis (Figure 2C and D). Accordingly, the combination of 5FU and andrographis significantly increased the percentage of late apoptotic cells in HCT116 5FUR cells (combination 25.85% versus 5FU 8.13%; P < 0.0001) and SW480 5FUR cells (combination 16.2% versus 5FU 8.48%; P = 0.0002). To further validate these findings, we analyzed the influence of andrographis on expression of caspase-9, a key player in the intrinsic apoptotic pathway. Gene expression analysis revealed a significant increase in its expression in combination group when compared with 5FU treatment in both HCT116 5FUR and SW480 5FUR cells lines (P < 0.05; Figure 2E), yet again highlighting the ability of andrographis to chemosensitize proapoptotic activity in otherwise chemoresistant colon cancer cells.

Andrographis-mediated chemosensitization in colon cancer is primarily through activation of ferroptosis and inhibition of β-catenin/Wnt-signaling pathways

Next, to decipher the most important molecular pathways responsible for the chemosensitizing properties of andrographis, we performed a genomewide transcriptomic profiling analysis in parental, chemoresistant and andrographis-treated HCT116 and SW480 cell lines. We were specifically interested in identifying genes that were differentially expressed between 5FU versus the combination treatment with andrographis. As expected, we observed a large number of genes that were significantly upregulated (n = 393) and downregulated (n = 382) between these two treatment groups. Gene set enrichment analysis on these significantly dysregulated genes identified led us to identify that ferroptosis and β-catenin/Wnt-signaling pathways were the two pathways that were significantly enriched and commonly dysregulated in both chemoresistant cell lines (Figure 3A). Further analysis of the specific genes within these two pathways revealed that within the ferroptosis pathway, HMOX1, GCLC, GCLM, FTL, SAT1, STEAP3, TP53 and ACSL5 were significantly upregulated, whereas within the β-catenin/Wnt-signaling pathway, WNT16, AXIN2, ROR2, FZD4, PPP3CA and TCF7L2 genes were frequently dysregulated following andrographis treatment (P < 0.05; Figure 3B).

Figure 3.

Genomewide microarray analysis for the identification of key pathways influenced by andrographis and 5FU. (A) Various cancer-associated pathways that were either activated or suppressed by andrographis when used with 5FU in 5FUR CRC cells when compared with only 5FU treatment. (B) Heatmap showing the expression of genes involved in ferroptosis and β-catenin/Wnt-signaling pathways in combination treatment when compared with only 5FU treatment in 5FUR CRC cells.

Considering that gene expression profiling results from microarray experiments must be validated by quantitative PCR assays and western immunoblotting, we analyzed these candidate genes using these assays. Interestingly, HMOX1, which is a critical mediator of ferroptosis induction, was the most upregulated gene (log₂ fold change = 8) in response to treatment with andrographis versus 5FU, in both cell lines. Further validation analysis using qRT–PCR assays confirmed a significant increase in the expression of several of the ferroptosis-related genes including HMOX1, GCLM and GCLC (Figure 4A). It was intriguing to observe that even the protein expression of HMOX1 and GCLM was significantly upregulated by andrographis individually and in combination in HCT116 and SW480 5FUR cells, whereas 5FU treatment by itself did not alter their expression (Figure 4C and D), highlighting the specific effects of andrographis in mediating ferroptosis in these cell lines. Likewise, the drug-resistant cells demonstrated decreased expression of WNT16, TCF7L2 and AXIN2 following treatment with andrographis versus 5FU alone, indicating the suppression of Wnt-signaling pathway (Figure 4B). In particular, TCF7L2, which is an effector molecule of β-catenin/Wnt pathway, has been suggested to have a significant role in chemoresistance and tumor recurrence (31,32). We noted that the expression of TCF7L2 was significantly downregulated by andrographis in 5FUR cell lines even at protein expression levels, whereas 5FU treatment alone did not have any impact on the expression of this gene (Figure 4C and D). Collectively, these results indicate that activation of ferroptosis and suppression of β-catenin/Wnt pathways are among the potential key mechanisms contributing toward chemosensitizing properties of andrographis.

Figure 4.

Andrographis inhibits β-catenin/Wnt-signaling pathways and activates ferroptosis-related genes in CRC cells. Expression of genes involved in (A) ferroptosis and (B) Wnt pathway in HCT116, SW480, HCT116 5FUR and SW480 5FUR cells treated with andrographis and/or 5FU when compared with vehicle control. Protein immunoblot analysis of HMOX1, GCLC, GCLM and TCF7L2 in (C) HCT116 5FUR and (D) SW480 5FUR cells treated with andrographis and/or 5FU.

Andrographis treatment resensitized 5FU-resistant CRC tumor growth in a xenograft animal model

To further confirm our findings from cell culture studies, we next established a xenograft animal model using HCT116 5FUR cells, to interrogate the effects of andrographis in its ability to induce resensitization of 5FU-resistant colon cancer. In these in vivo experiments, the animals received 5FU and andrographis treatment through intraperitoneal injections, either individually or the combination of two compounds, as illustrated in the study design in Figure 5A. We observed that although the tumors continued to grow in mice that were administered 5FU alone, coadministration of andrographis significantly attenuated tumor volume (P < 0.001; Figure 5B and C) and weight (P < 0.001; Figure 5D), highlighting the anti-cancer activity of andrographis in this animal model.

Figure 5.

Andrographis enhances sensitivity to 5FU in HCT116 5FUR cells in a xenograft animal model. (A) Schematic for the generation of xenograft model in athymic mice and the treatment groups. (B) An image illustrating xenograft tumors harvested from various treatment groups. (C) A line plot depicting changes in progressive tumor volume with various treatments. (D) Changes in tumor weights in various treatment groups. qRT–PCR analysis of genes involved in (E) ferroptosis and (F) Wnt pathway in tumors treated with 5FU and the combination. Normalization was performed using β-actin, and fold change was determined when compared with group treated with vehicle. HCT116 5FUR, 5FU-resistant HCT116 cell line. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

In addition, in line with our in vitro results, coadministration of andrographis along with 5FU enhanced the expression of key ferroptosis pathway genes including HMOX1, GCLC and GCLM, which was significantly more effective compared with 5FU treatment individually (Figure 5E). Similar observations were made for the significantly reduced expression of β-catenin/Wnt-signaling pathway genes WNT16 and TCF7L2 in the xenograft tumor tissues that were derived from animals treated with the combination of andrographis with 5FU (Figure 5F). The human equivalent dose of andrographis would equate to about 600 mg/day for a 60 kg human, which is quite feasible as a dietary supplement as well. Collectively, our xenograft animal model experiments validated the results from in vitro experiments and highlight the anti-cancer and chemosensitizing effects of andrographis in colon cancer, primarily through the dysregulation of ferroptosis and β-catenin/Wnt-signaling pathways.

Andrographis treatment resulted in significant inhibition of tumor growth in patient-derived organoids

Finally, we asked whether the effect of andrographis treatment can also be confirmed in ex vivo tumors derived from patients with CRC. To answer this question, we generated patient-derived tumor organoids from two CRC patients and examined the effects of andrographis. Organoid cultures derived from human CRC tissues were treated with 5FU, andrographis and their combination for a week (Figure 6A). In line with our results from the cell lines and xenograft animal model, the combination treatment with andrographis significantly decreased the growth of patient-derived tumor organoid formation vis-à-vis 5FU treatment alone (P = 0.0061 for patient 1 and P < 0.05 for patient 2; Figure 6B). Furthermore, consistent with our results from in vitro and in vivo studies, we were able to successfully confirm the activation of the expression of key ferroptosis-related genes, GCLC, GCLM and HMOX1, even in the tumor organoids following treatment with andrographis versus 5FU alone (Figure 6C), highlighting that combined treatment with andrographis was significantly more effective in inhibiting organoid growth compared with either compound individually.

Figure 6.

A combination of andrographis and 5FU effectively suppressed growth of patient-derived CRC organoids. (A) Representative images showing tumor organoid cultures derived from two different CRC patients, treated with vehicle, 5FU, andrographis and combination of the two. Representative arrows indicate different sizes of organoids considered for counting. (B) Bar graphs illustrate significant decrease in organoid count after andrographis treatment. (C) Gene expression changes in ferroptosis-related genes in patient-derived organoids treated with 5FU and andrographis in combination or individually. mRNA levels of β-actin were used as the internal normalizing control. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

CRC represents one of the most common cancers in USA, and the lack of effective treatments for this disease, has inspired both researchers and clinicians to identify all potential treatment approaches that can improve survival and therapeutic outcomes in this malignancy. In view of the de novo and acquired chemoresistance to the standard 5FU-based chemotherapeutic regimen in CRC, and the growing body of evidence that active principles in various naturally occurring botanicals might help overcome such resistance, we undertook this study to interrogate the effects of andrographis in its ability to induce chemosensitization in CRC. To answer this question, we performed a series of systematic studies in cell culture, animal model and patient-derived tumor organoids and provided a previously unrecognized effects of andrographis in mediating chemosensitization in cancer cells through the activation of ferroptosis and inhibition of β-catenin/Wnt-signaling pathways.

The chemotherapeutic drug, 5FU, is widely used for the treatment of many types of cancers, including CRC. However, a serious clinical limitation of 5FU-based treatment is that >50% of patients exhibit resistance to this treatment modality (4,33), highlighting the need for further improvization in overcoming such chemoresistance and improving overall treatment outcomes in cancer patients. Numerous studies in the last decade have shown great promise that active principles in various nutraceuticals such as curcumin, grapeseed extract and boswellia help resensitize cancer cells to 5FU-based drugs (30,34–37), such evidence in the context of andrographis remains largely unclear (24,38). To fill this important gap in knowledge, our present study is important, as we provided novel evidence in this regard on multiple levels whereby we demonstrate that andrographis extract potentiates the effects of 5FU in chemoresistant CRC cell lines previously established in our laboratory (37), in a xenograft animal model and two independent patient-derived tumors. It was interesting to observe that although the individual IC₅₀ concentrations for 5FU were never achieved in 5FUR cells, cotreatment with andrographis significantly reduced the IC₅₀ concentrations for 5FU in chemoresistant cell lines, as was the case in the parental cells. Interestingly, androgrphis was effective in both MMR-deficient and MMR-proficient cell lines. Although MMR-deficient CRC could benefit from immune checkpoint blockade therapy due to high expression levels of checkpoint proteins in the local immune microenvironment (39) and the strong T-cell response induced by large amounts of neoantigens (40), almost 90% of CRCs are MMR proficient and lack adequate therapeutic options (41). In this context, use of andrographis, which is safe and inexpensive nutraceutical in MMR-proficient CRCs, might be an attractive option for such patients. Moreover, andrographis alone appeared to have high efficacy in suppressing tumor volume in xenograft assay. It could be due the multitargeting properties of andrographolide. Multiple signaling pathways, including Wnt/β-catenin-signaling pathway, ERK1/2 and NF-κB, JAK2 transducers and PI3K pathway, have been associated with the pharmacological activity of andrographolide (42,43). It has been demonstrated that A.paniculata extract or andrographolide could stimulate cytotoxic T lymphocyte production through the enhanced secretion of interleukin-2 and interferon-γ by T cells, thereby inhibiting tumor growth in vivo. Inhibition of angiogenesis is currently perceived as a promising strategy in treating cancer (43). Likewise, both A.paniculata extract and andrographolide were found to inhibit tumor-specific angiogenesis by regulating the production of various pro- and anti-angiogenic factors, such as proinflammatory cytokines, NO, vascular endothelial growth factor, interleukin-2 and the tissue inhibitor of metalloproteinase-1 (44,45). Taken together these reports highlight the potency of andrographis treatment alone in suppressing tumor growth however further validation in clinical trials is warranted.

From a mechanistic standpoint, we performed genomewide expression analysis in paired parental and 5FUR cell lines treated with both compounds and identified that andrographis exerted its efficacy through activation of the ferroptosis pathway and inhibition of β-catenin/Wnt-signaling pathways in chemoresistant cells. Ferroptosis is a process of programmed necrosis, which is mainly triggered by extra-mitochondrial lipid peroxidation arising from an iron-dependent reactive oxygen species accretion. Recent studies have shown that the induction of ferroptosis sensitizes resistant cells to cisplatin in human malignancies (46,47). Increased expression of HMOX1 was an important event in erastin-induced ferroptosis in HT-1080 fibrosarcoma cells and catalyzed the degradation of heme to ferrous iron, biliverdin and carbon monoxide, enhancing ferroptosis by increasing hydroxyl radical (·OH) and non-enzymatic lipid peroxidation (48,49). Our present study demonstrated that andrographis-mediated chemosensitization in CRC cells to 5FU was also in part due to the enhanced expression of HMOX1 and GCLM genes, supporting the previously reported anti-cancer and anti-inflammatory properties of andrographis in cancer (50,51). It is likely that the specific upregulation of HMOX1 and GCLM by andrographis might be through altered PI3K/Akt pathway, involving Nrf2 and AP-1 transcriptional factors, as has been reported previously (52).

The other potential mechanism for the anti-cancer and chemosensitizing effects of andrographis observed in this study was suppression of β-catenin/Wnt-signaling pathway, through the downregulation of Wnt16, AXIN2 and TCF7L2 genes. The β-catenin/Wnt-signaling pathway hyperactivation is arguably the most critical cancer driver and has been shown to be involved in 5FU-mediated drug resistance in CRC cells (53). The TCF7L2 protein (also known as TCF4) is an important regulator of canonical Wnt/β-catenin signaling in Wnt-activated cells. Interestingly, TCF7L2 was shown to be frequently overexpressed in rectal cancers resistant to preoperative 5FU-based long-term chemoradiotherapy (54), as well as shRNA-mediated silencing of this gene sensitized CRC cell lines to clinically relevant doses of 5FU and radiation (31,55). Because it has been a priority to generate safe and effective molecules targeting β-catenin/Wnt-signaling pathway in CRC to overcome chemoresistance, given the safety and affordability of andrographis offers an attractive option for its potential use as an adjunctive treatment along with conventional chemotherapeutic drugs.

In conclusion, herein, we provide a previously unrecognized effect of andrographis in its ability to mediate chemosensitization in CRC, in part through the activation of ferroptosis pathway and downregulation of β-catenin/Wnt-signaling pathways. Therapeutic targeting of these pathways has long been recognized as a promising approach for overcoming chemoresistance in cancer, and our present study provides an important evidence for the potential use of andrographis as an adjunctive treatment to 5FU-based chemotherapy, for improving the overall treatment outcomes in patients suffering from CRC.

Abbreviations

Abbreviations
 
• 5-FU

  5-fluorouracil

 
• CI

  combination index

 
• CRC

  colorectal cancer

Funding

This work was supported by CA184792, CA187956, CA227602, CA072851 and CA202797 grants from the National Cancer Institute, National Institutes of Health.

Author contributions

Concept and design: PS, TS, JB and AG; acquisition, analysis or interpretation of data: PS, TS and JB; drafting of the manuscript: PS, TS, JB and AG; statistical analysis: PS, TS and JB; Administrative, technical, or material support: PS, TS, JB and AG; supervision: AG.

Conflict of Interest Statement: None declared.