Novel strategies to reverse chemoresistance in colorectal cancer

Abstract Colorectal cancer (CRC) is a common gastrointestinal malignancy with high morbidity and fatality. Chemotherapy, as traditional therapy for CRC, has exerted well antitumor effect and greatly improved the survival of CRC patients. Nevertheless, chemoresistance is one of the major problems during chemotherapy for CRC and significantly limits the efficacy of the treatment and influences the prognosis of patients. To overcome chemoresistance in CRC, many strategies are being investigated. Here, we review the common and novel measures to combat the resistance, including drug repurposing (nonsteroidal anti‐inflammatory drugs, metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and S‐adenosylmethionine), gene therapy (ribozymes, RNAi, CRISPR/Cas9, epigenetic therapy, antisense oligonucleotides, and noncoding RNAs), protein inhibitor (EFGR inhibitor, S1PR2 inhibitor, and DNA methyltransferase inhibitor), natural herbal compounds (polyphenols, terpenoids, quinones, alkaloids, and sterols), new drug delivery system (nanocarriers, liposomes, exosomes, and hydrogels), and combination therapy. These common or novel strategies for the reversal of chemoresistance promise to improve the treatment of CRC.


| INTRODUCTION
Colorectal cancer (CRC) is the third most prevalent cancer around the world, with increasing morbidity and mortality every year. 1 As reported by the International Agency for Research on Cancer (IARC) in 2020, there were over 193,000 new cases of CRC and more than 953,000 deaths worldwide, and CRC accounted for 10.0% of overall cancer incidence. 2 Currently, the treatment of CRC mainly consists of surgery, targeted therapy, radiotherapy, and chemotherapy. 3 Generally, chemotherapy, as the most classical and most common-used therapy in CRC, can be applied at different stages of the treatment and is commonly provided after surgery as adjuvant therapy for patients with advanced CRC. 4 Treatment with chemotherapeutic agents like 5-fluorouracil (5-FU), oxaliplatin (L-OHP), vincristine (VCR), doxorubicin (DOX), cisplatin (CDDP)and irinotecan (CPT-11) has improved the overall survival of patients with advanced CRC in the past decades. 5 However, even though chemotherapy is well demonstrated to reduce tumor burden and prolong survival, it remains a palliative treatment since most CRC patients eventually exhibit drug resistance. 6 More than 90% of patients with metastatic cancer may suffer chemotherapy failure due to drug resistance. 7 Chemotherapy resistance, according to drug responsiveness, can be classified into two types: intrinsic resistance and acquired resistance. It can also be divided into primary drug resistance and multidrug resistance (MDR) in terms of the drug resistance spectrum. Multidrug resistance is seem to be the major determinant of cancer chemotherapy failure. Nowadays, drug resistance not only undermines the therapeutic effects of anticancer chemical drugs but also causes CRC refractory. Therefore, drug resistance has become a thorny issue and urgently needed to be solved. Various strategies have been applied or investigated to the drug resistance reversal in CRC. The most common treatment among them is the combined use of chemical drugs and marketed medicines with already known effects such as drug repurposing (nonsteroidal anti-inflammatory drugs, metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and Sadenosylmethionine) and natural herbal compounds (polyphenols, terpenoids, quinones, alkaloids, and sterols). Gene therapy, as an emerging technology, has also been studied in chemoresistance reversal, including ribozymes, RNA interference, CRISPR/Cas9, epigenetic therapy, antisense oligonucleotides, and noncoding RNAs, which opens the door to a new genetical world in this field. Protein inhibitors are countermeasures at protein levels, which exserts their reversal effects directly by reacting with specific proteins such as EGFR inhibitor, S1PR2 inhibitor, and DNA methyltransferase inhibitor. It has been known that conventional drug delivery systems have many shortcomings comprising low bioavailability and cytotoxicity, which contribute to drug resistance. New drug delivery systems, including nanocarriers, liposomes, exosomes, and hydrogels, can overcome the drawbacks of traditional delivery systems and transport the therapeutic molecules directly to the specific tumor site, which plays an important role in reversing chemoresistance ( Figure 1).
In this review, the aforementioned novel strategies to overcome chemoresistance in CRC will be clarified, which will provide new ideas for the treatment of drug resistance in CRC.

| DRUG REPURPOSING
Recently, increased classical drugs, which were widely used in the clinic for different diseases, have been found to possess the ability to reverse chemoresistance in CRC, such as some nonsteroidal anti-inflammatory drugs (aspirin, ibuprofen, and NS-398), metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and S-adenosylmethionine. Due to their low expenses, low toxicity, and high safety, drug repurposing is a promising strategy for combating chemoresistance in CRC (Table 1).

| Nonsteroidal anti-inflammatory drugs
Nonsteroidal anti-inflammatory drugs (NSAIDs), as a kind of widely used medicine worldwide, have potent anti-inflammatory, analgesic, and antipyretic activity. The main mechanism of the action is the inhibition of cyclooxygenase (COX), which is responsible for the biosynthesis of prostaglandins and thromboxane. 8 Aspirin, ibuprofen, and NS-398 are found to own the potential for overcoming drug resistance in CRC. It was reported that the combined use of aspirin (a nonselective COX inhibitor) and 5-FU could reverse drug resistance and potentiate the antineoplasm effect of 5-FU by abolishing 5-FU-induced nuclear factor-kappaB (NF-κB) activation in 5-FU-resistant SW480, SW620 (SW480/5-FU and SW620/5-FU) cell lines and xenograft mice of CRC. 9 Wawro et al. found that both aspirin and ibuprofen inhibit the VCR-dependent secretion of tumor growth factor-βs (TGF-βs) and interleukin-6 (IL-6) from cancer-associated fibroblasts (CAFs) in CAFlike cells, suggesting that aspirin and ibuprofen may reverse VCR-resistance in CRC to some extent. 10 NS-398, a selective COX-2 inhibitor, could significantly inhibit the expression of multidrug resistance protein 1 (MDR1) mRNA, P-glycoprotein (P-gp/ABCB1), phosphorylated-c-Jun levels and increase the intracellular concentration of VCR in VCR-resistant HCT-8/VCR cell line, promoting the sensitivity to VCR in resistant cells. 11

| Metformin
Metformin (MET), which suppresses hepatic glucose production, is the first-line therapy for type 2 diabetes mellitus. 12 MET has been recently demonstrated to reverse the resistance after chemotherapy and possess a potential therapeutic effect on colorectal cancer. 13 MET enhanced the chemosensitivity of SW480 and SW620 cell lines to CDDP, inhibited cell proliferation, induced cell apoptosis, and increased the production of reactive oxygen species (ROS) through ROS-mediated phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway. 14 MET was also found to sensitize HCT1116 and SW480 cell lines to CPT-11-induced cytotoxicity and block the cell cycle in G1 and S phases, which indicates an innovative strategy in the treatment of CPT-11-resistant CRC. 15 Moreover, MET could inhibit cell proliferation, migration, and cancer stem cell (CSC) population in 5-FU-resistant SNU-C5 CRC cell line, which was mediated by the activation of AMP-activated protein kinase (AMPK) and suppression of DNA replication, and NF-κB pathway. 16 MET was also found to resensitize HCT116 cells to 5-FU resistance, which was realized by attenuating stemness and epithelial-mesenchymal transformation (EMT) via inhibiting the Wnt3a/β-catenin pathway. 17 Furthermore, it was found that the combined use of MET and propranolol (mainly used for hypertension treatment) could block 5-FU chemoresistance in HCT116 cells to some extent, which could be applied as a putative adjuvant treatment for CRC and chemo-resistant CRC. 18

| Enalapril
Enalapril, an antihypertensive and antiheart failure drug, was found to inhibit cell migration and invasion and overcome chemoresistance in the 5-FU-resistant CRC cells at clinically acceptable dosages without extra toxicity. And the effect may be primarily achieved by the suppression of cell proliferation, angiogenesis, and NF-κB/signal transducer and activator of transcription 3 (STAT3)-regulated proteins, including Cyclin D1, c-Myc, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), Bcl-2, and X-linked inhibitor of apoptosis protein (XIAP). 21

| Ivermectin
Ivermectin, an antiparasitic drug, could reverse the chemoresistance in VCR-resistant HCT-8/VCR cells by

| Bazedoxifene
Bazedoxifene, the third-generation of selective estrogen receptor modulator, also a novel IL-6/glycoprotein 130 (GP130) target inhibitor, improved the anticancer effect of 5-FU in CRC via impeding the IL-6/GP130 signaling pathway and the phosphorylation of the downstream effectors such as AKT, ERK, and STAT3, suggesting that the block of IL-6/GP130 might reverse chemoresistance in CRC. 23

| S-adenosylmethionine
S-adenosylmethionine (AdoMet), a natural chemical and generally utilized as a dietary supplement, was capable of conquering 5-FU chemoresistance in CRC cells through reverting the P-gp upregulation induced by 5-FU and suppressing the activation of the key anti-apoptotic factor NF-κB implicated in P-gp-related chemoresistance. 25

| Vitamins
Vitamins are common dietary supplement used in daily life and their potential effects on chemoresistance reversal in CRC have been found, including vitamin C and vitamin D. Vitamin C, also called ascorbic acid, could sensitize the human CRC cells to L-OHP by inducing cell apoptosis. 26 Vitamin D, which promotes calcium absorption and maintains bones healthy, could increase the sensitivity of human CRC cells to 5-FU by suppressing TYMS and survivin expression in a calcium-sensing receptor (CaSR) -dependent manner. 27 Taghizadeh et al. found that the active metabolite of vitamin D 1α,25-dihydroxy vitamin D3 (1,25-D3) could restore the sensitivity to 5-FU and CPT-11 in 5-FU/CPT-11-resistant MIP101 human CRC cell line. Furthermore, they also found that combined 1α,25-dihydroxy vitamin D3 with secreted protein acidic and rich in cysteine (SPARC) which is a matricellular protein could augment chemosensitivity in CRC through upregulating the expression of vitamin D receptor (VDR) with a lower dosage of chemo drugs. 28

| Ribozymes
Ribozymes are small RNA molecules with catalytic functions, which can degrade specific mRNA sequences. Transfection of the anti-MDR1 ribozyme, bound to the carcino-embryonic-antigen (CEA) promoter, could reverse DOX resistance by reducing P-gp expression in SW1116R MDR CRC cells. 30 Hammerhead ribozyme-mediated-specific suppression of MDR1 and multidrug resistance-associated protein (MRP) could reverse resistance to DOX and etoposide (VP-16) in CDDP-resistant HCT-8/CDDP cells. 31 Hammerhead ribozyme against γglutamylcysteine synthetase (γ-GCS) could reverse resistance to CDDP, DOX, and VP-16 in CDDP-resistant HCT-8 cells by markedly downregulating the expression of the γ-GCS gene and MRP/MDR1. 32

| Antisense oligonucleotides
Antisense oligonucleotides (ASOs) are synthesized nucleic acids, containing 12-25 nucleotides in general, which exert their inhibitive effect by binding to the target RNA through Watson-Crick hybridization. 33 It was reported that combined use of miR-21 inhibitor oligonucleotide (miR-21i) and 5-FU delivered by the engineered exosomes could overcome resistance and markedly enhance the drug cytotoxicity in 5-FU-resistant HCT-116/5-FU cell line by increasing apoptosis, inducing cell cycle arrest, reducing tumor proliferation, and rescuing the decrease of phosphatase and tensin homolog (PTEN) and hMSH2 (regulatory targets of miR-21) due to the downregulation of miR-21. 34 The antisense oligonucleotide of miR-19a (anti-miR-19a) has been found to reverse L-OHP resistance in L-OHP-resistant SW480 and HT29 cell lines via PTEN /PI3K/AKT pathway. 35

| RNA interference
RNA interference (RNAi) regulates gene expression and contributes to the targeted therapy through 20-24 bp RNA including small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs).

| Short hairpin RNAs
A disintegrin and metalloproteinase 17 (ADAM17) shRNA could inhibit cell proliferation, increase apoptosis, and reverse L-OHP resistance in L-OHP resistant HCT-8 cell line by suppressing the EGFR/PI3K/AKT signaling pathway. 41 Silencing PIK3CA and PIK3CB by RNAi could repress the capability of proliferation, migration, and invasion of CRC cells and reverse MDR in 5-FU-resistant HCT-8/5-FU cell line. 42 GOLPH3 shRNA could reverse L-OHP resistance by limiting cell proliferation and inducing apoptosis through the suppression of P13K/AKT/mTOR pathway in L-OHP resistance HCT116/L-OHP cell line. 43 RNAi of CD147 was observed to inhibit cell proliferation and invasion and resensitize HT29 cells to CDDP and DOX. 44 Transfected of livin (an inhibitor of apoptosis proteins) shRNA to VCR resistant HCT-8/VCR cells could increase apoptosis in response to VCR, VP-16, and 5-FU, which is able to reverse drug resistance. 45 RNAi of carboxylesterase-2 can suppress proliferation and induce apoptosis and reverse L-OHP resistance by suppression of PI3K signaling in L-OHPresistant HCT116/L-OHP and RKO/L-OHP cell lines. 46 Overexpression of nuclear protein FK506-binding protein 3 (FKBP3) and histone deacetylase 2 (HDAC2) could promote L-OHP resistance in CRC cells, whereas knockdown of FKBP3 could abate L-OHP resistance by decreasing HDAC2 expression and perhaps through regulating PTEN/AKT pathway in CRC cells. 47 Knockdown of CDK2-associated cullin domain 1 (CAC1), an innovative regulator of cell cycle, could promote the sensitivity of SW480/5-FU and LoVo/5-FU cell lines to 5-FU by inducing cell apoptosis, arresting tumor cells at the G1/S phase, and lowering the expression of P-gp and MRP-1. 48 Knockdown of CSC biomarker CD133 could reverse DOX chemoresistance by downregulating MDR1/P-gp expression via AKT/NF-κB/MDR1 signaling in DOXresistant LoVo and HCT8 cell lines. 49 Knockout of SLC25A22 increased chemosensitivity of KRAS-mutant DLD1 and SW1116 cell lines to 5-FU, and it is also affirmed in DLD1 xenograft mice. 50

| CRISPR/Cas9
CRISPR/Cas9 technology, a powerful, efficient, easy, and specific gene editing tool, is extensively administrated in tumor pathogenesis, treatment, and drug resistance by inserting or knocking out the related genes. 51 Identifying and modulating resistance-related genes by CRISPR/Cas9 is an efficient way to reverse chemoresistance and inhibit tumor recurrence. Knockout ABCB1 gene by CRISPR/Cas9 tool increased the chemosensitivity of ABCB1 overexpressed MDR HCT8/VCR cell line to DOX by increasing intracellular accumulation of DOX. 52 Similarly, Lei et al. also found that ABCB1 knockout could reverse 3H-paclitaxel (PTX) resistance mediated by ABCB1 in SW620/Ad300 CRC cells by decreasing drug efflux. 53 Knockout of redox-inducible antioxidant protein RING-box 2 (RBX2) by CRISPR/Cas9 technique enhanced the sensitivity of HCT116 and SW480 cell lines to PTX probably by deactivating mTOR/S6 kinase 1 (S6K1). 54 Knockdown of T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1) enhanced sensitivity to 5-FU and reduced tumor invasiveness in HCT116 xenograft mice. 55

| Micro-RNAs
Micro-RNAs (MiRNAs), defined as small ncRNAs containing 21-23 nucleotides, regulate gene expression at the level of posttranscription by repressing mRNA translation and inducing mRNA degradation. 57 Dysregulation of miRNAs is associated with colorectal carcinogenesis, and appropriate modulation of miRNAs is found to exert reversal effects in resistant CRC cells. 58,59 Wu et al. found that upregulation of miR-200b-3p could promote cell migration, induce apoptosis, inhibit cell growth, and reverse L-OHP resistance by downregulating the expression of βIIItubulin protein through binding to 3′-UTR of TUBB3 (encoding β III-tubulin protein) in L-OHP-resistant HT29/L-OHP and HCT116/L-OHP cell lines. 60 Zhou et al.
reported that overexpression of miR-506 improved chemosensitivity in HCT116/L-OHP cell line by suppressing P-gp expression through downregulating Wnt/β-catenin pathway. 61 Sun et al. indicated that miR-27b-3p could reverse chemoresistance by inhibiting autophagy via inhibiting the expression of autophagy-related protein ATG10 in L-OHP-resistant SW480 cells. 62 MiR-195-5p could dramatically increase chemosensitivity in 5-FU-resistant HCT116/5-FU and SW480/5-FU CRC cell lines by reducing the expression of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) that is involved in the process of choline phospholipid metabolism. 63 MiR-133b reduced stemness and 5-FU and L-OHP chemoresistance in HT29 and SW620 cell lines by targeting and suppressing the disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79me2 modification and transcription of stem cell genes. 64 MiR-375-3p was discovered to be downregulated in CRC tissues as well as HT29 and HCT116 cell lines. It strengthened the sensitivity to 5-FU in CRC cells by inducing cell cycle arrest and apoptosis via targeting TYMS. 65 Upregulation of miR-1914* and miR-1915 decreased chemoresistance in 5-FU and L-OHP chemo-resistant HCT116 cells by repressing the expression of transcription factor nuclear factor I/X (NFIX). 66 MiR-138-5p, which decreased in CRC tissues, could inhibit cell migration and chemoresistance in CRC by targeting the nuclear factor I/B (NFIB)-Snail1 axis. 67 Combined with 5-FU and L-OHP, miR-193a-5p reversed chemoresistance in CRC by reducing C-X-C motif chemokine receptor 4 (CXCR4, induces cell proliferation and metastasis) expression. 68 MiR-195-5p considerably increased tumor cell apoptosis, reduced tumor sphere formation, and prevented cell stemness and chemoresistance by decreasing Notch signaling proteins Notch2 and recombination signal binding protein for immunoglobulin Kappa J region (RBPJ). 69 Suppression of miR-135b and miR-182 may reverse the 5-FU resistance in HCT-8/5-FU and LoVo/5-FU cell lines by targeting sialyltransferase ST6GALNAC2 via PI3K/AKT pathway. 70 Overexpression of miR-26b could reverse 5-FU chemoresistance by downregulation of P-gp in 5-FU-resistant HT-2 and LOVO cell lines. 71

| Long noncoding RNAs
Long noncoding RNAs (LncRNAs), which are transcripts that exceed 200 nucleotides, can regulate CRC progression by acting as competitive endogenous RNAs (ceR-NAs) by interacting with miRNAs or protein-coding mRNAs and encoding short peptides. 72 A growing number of studies has indicated that abnormally expressed lncRNAs confer to drug resistance in CRC and the condition can be overcome by re-modulating the expression of the aberrant lncRNAs. Downregulation of lncRNA colon cancer-associated transcript 1 (CCAT1) could effectively inhibit CCAT1 expression and reverse the 5-FU resistance in HCT116/5-FU and HT29/5-FU cells. 73 Long intergenic nonprotein-coding RNA 689 (LINC00689) functioned as a miR-31-5p sponge to inhibit tumor proliferation, metastasis, and chemoresistance by upregulating large tumor suppressor kinase (LATS2) and repressing yes-associated protein (YAP)/β-catenin signaling pathway in CRC. 74 LncRNA MIR600HG functioned as a tumor suppressor and the overexpression of MIR600HG could inhibit tumor invasion and enhance chemosensitivity by targeting ALDH1A3 (encodes an aldehyde dehydrogenase enzyme) in CRC. 75 LncRNA lnc-AP could sensitize the HCT116/L-OHP and SW480/L-OHP cell lines to L-OHP by encoding short peptide pep-AP, which suppressed pentose phosphate pathway (PPP), increased ROS accumulation and cell apoptosis through inhibiting transaldolase 1 (TALDO1, a key enzyme of PPP). 76

| Epigenetic therapy
Epigenetic therapy is a coping strategy to modify the aberrant epigenetic alterations in cancers, including CRC. It has been found that epigenetic alterations like higher DNA methylation and mRNA N6-methyladenosine (m6A) are related to the occurrence of chemoresistance and tumor relapse in CRC. 86 Block the epigenetic modification that occurred in resistant CRC can reverse the resistance. Baharudin et al. found that recurrent CRC patients exhibited higher methylation levels and the recurrence of CRC compared to non-recurrent CRC patients, which might associate with the abnormal methylation of CCNEI, CCNDBP1, CHL1, DDX43, and PON3. Treatment of 5-aza-2′-deoxycytidine (5-azadC, a DNA methylation inhibitor) could restore the sensitivity to 5-FU in the SW48 cell line. 87 Zebularine, a low-toxicity DNA methyltransferase (DNMT) inhibitor, could overcome hypoxia-induced CDDP resistance in HCT116 cells and show the same efficacy in HCT116 xenograft models and AOM/DSS-induced CRC mouse models by downregulating HIF-1α expression through hydroxylation. 88 Zhang et al. found that methyltransferase-like 3 (METTL3) and mRNA N6-methyladenosine (m6A) were upregulated in 5-FU-resistant HCT-116 and SW480 cell lines.
Knockdown of METTL3 could restore the chemosensitivity of HCT-116/5-FU, SW480/5-FU, and SW620/5-FU cell lines mediated by downregulating the expression of lactate dehydrogenase (LDH) A, which catalyzes pyruvate to lactate to promote glycolysis. 89 Uddin et al. found that silencing of METTL3 or inhibition of RNA methylation could restore the sensitivity to DOX in cells carrying the mutation of R273H by suppressing the m6A modification in the pre-mRNA of p53 and increasing the phosphorylated level of p53 protein. In addition, suppression of ceramide glycosylation was also found to combat drug resistance by mechanisms of the suppression of METTL3 and m6A formation in p53 pre-mRNA. 90 Overexpression of methyltransferase-like 1 (METTL1) was found to sensitize CDDP-resistant colon cancer cells to chemoresistance via modulating miR-149-3p/S100A4/p53 axis. 91 Wojtowicz et al. found that tunicamycin, an inhibitor of N-glycosylation, might reverse MDR in LoVo/DOX cell line by blocking the first-step N-glycosylation and translocation of P-gp. 92

| PROTEIN INHIBITORS
In the treatment of CRC chemoresistance, except for gene-level therapy, direct protein inhibitors also play an important role. Various inhibitors, including inhibitors of classical resistance-related proteins, EGFR inhibitors, and sphingosine 1-phosphate receptor (S1PR) modulators have been found to reverse chemoresistance in CRC.

| Inhibitors of classical resistancerelated proteins
An increment in drug efflux could reduce drug concentration and accumulation in tumor cells, which leads to drug resistance. Targeting ATP-binding cassette (ABC) transporters like breast cancer resistance protein (BCRP) and P-gp provides a potential approach to eliminating drug resistance during the treatment of CRC. GF120918, a potent P-gp and BCRP inhibitor, markedly increased the oral bioavailability of topotecan from 40% to 97% in cancer patients. 93 Compound WS-10, a P-gp inhibitor, could overcome P-gp-mediated MDR in SW620/Ad300 cells by binding with P-gp, thus enhancing the intracellular accumulation of PTX. 94 The protein kinase C (PKC) family, deemed as oncoprotein historically, is comprised of a group of serine or threonine kinases and is incorporated in tumor progression. 95 It has been indicated that PKC could reduce intracellular drug concentration by activating ATP-dependent efflux pumps, which contributes to drug resistance in CRC. 96 Go6976, a specific inhibitor of classical PKC, can be used to reverse DOX resistance in human colon cancer DOX-resistant HCT15 cell line by reducing MDR expression and increasing DOX-induced apoptosis. 97

| EGFR Inhibitors
EGFR is a transmembrane receptor belonging to a family of receptor tyrosine kinases that mediates a series of intracellular pathways that promote tumor proliferation, invasion, metastasis, and neovascularization. 98 EGFR inhibitors application has been found to overcome chemoresistance in CRC. Saptinib, an EFGR inhibitor, could increase 3H-PTX accumulation in tumor cells and reverse MDR in SW720/Ad300 cells through stimulating ATPase which competitively inhibits 3H-PTX uptake. 99 Erlotinib, a specific inhibitor of EGFR, could effectively strengthen the antitumor effect of 5-FU via modulating the EGFR-FGD5-AS1-miR-330-3p-hexokinase 2 (HK2) pathway. 100

| S1PR modulators
Sphingosine-1-phosphate (S1P), one of the sphingolipids, could regulate cancer survival by binding sphingosine 1-phosphate receptors (S1PRs), which activate multiple cell growth-related pathways. 101 Inhibition of S1PR has been found to reverse chemoresistance in several cancers including CRC. JTE-013, an S1P and S1PR2 inhibitor, could overcome 5-FU resistance in CRC by lowering the expression of dihydropyrimidine dehydrogenase (DPD or DPYD). 102 S1PR2 antagonists compound 40 could markedly reverse the drug resistance in HCT116/5-FU and SW620/5-FU cell lines by evading the expression of dihydropyrimidine dehydrogenase (DPD). It significantly enhanced the inhibitory effect of 5-FU in the SW620/5-FU cells xenograft murine model without significant liver toxicity. 103

| Others
Notch and Wnt signaling-associated proteins were upregulated in HCT116/5-FU and HCT116/L-OHP cells, including Notch1 receptor intracellular domain NICD1 and nonphosphorylated βcatenin and Notch target gene HES1. Wnt inhibitor XAV939 and Notch inhibitor RO4929097 could restore cell viability in L-OHP-treated HCT116 cells. 104 Regorafenib, a multikinase inhibitor, which targets the RAS/RAF/ MEK/ERK pathway, could overcome the ABCB1mediated MDR and increase 3H-PTX accumulation in ABCB1-overexpressing tumor cells by suppressing the efflux activity of ABCB1. 105

| NATURAL HERBAL COMPOUNDS
Natural herbal compounds have become putative adjuncts of conventional chemotherapy in CRC and are able to reverse acquired drug resistance by different mechanisms. 106 Natural herbal compounds can be classified into polyphenols, terpenoids, quinones, alkaloids, sterols, and others according to the chemical structures (Table 3).

| Polyphenols
Dihydromyricetin, a flavonoid compound (belonging to polyphenols) extracted from the Japanese raisin tree (Hovenia dulcis), could inhibit both MRP2 expression and its promoter activity by inhibiting NF-κB-Nrf2 signaling in HCT116/L-OHP and HCT-8/VCR cell lines, contributing to the reversal of L-OHP/VCR-resistant CRC cells. 107 Curcumin, as a lipophilic polyphenol, was effective in the inhibition of cell proliferation, increment of cell apoptosis, block of G0/G1 phase, and downregulation of 10 and 11 translocation (TET1, a DNA demethylase), and naked cuticle homolog 2 (NKD2, a negative regulator of Wnt signaling), suggesting that curcumin might exert antiresistance effect 5-FU-resistant HCT116 cells by modulating the TET1-NKD2-Wnt signaling pathway, thus inhibiting the EMT progress. 108 Moreover, Fan et al. found that curcumin could evade tumor progression and reverse MDR in the HCT-8/5-FU cells line by suppressing the expression of P-gp and heat shock protein-27. 109 Resveratrol, a naturally occurring polyphenol, could induce chemosensitization to 5-FU in HCT116/5-FU and SW480/5-FU cell lines by inhibiting EMT via enhancing intercellular junctions and suppressing NF-κB pathway. 110 Resveratrol could also reverse L-OHP resistance by increasing drug accumulation, inhibiting MDR1, NF-κB pathway, and the transcriptional activity of cAMP-responsive elements in L-OHP-resistant HCT116 cells. 111

| Quinones
Emodin, a natural anthraquinone derivative, could reverse 5-FU resistance in SW480/5-FU cells by blocking cell proliferation, invasion, and migration as well as increasing cell apoptosis and downregulating the PI3K/ Akt pathway. 114 Tanshinones, diterpene quinones isolated from the roots of Salviamiltiorrhiza bunge, owe the properties of antioxidation, anti-inflammation, antitumor, and other pharmacological effects, and its chemoresistance reversal effect has been found in recent years. 115 Tanshinone IIA, cryptotanshinone, and dihydrotanshinone are three of the compounds of tanshinones, which reverse chemoresistance to some extent in CRC. Tanshinone IIA could combat L-OHP resistance in the SW480/L-OHP cell line by the inhibition of the ERK/Akt signaling pathway. Tanshinone IIA applied with L-OHP could significantly downregulate the expressions of Bcl-2, p-ERK, and p-Akt, and upregulate the expressions of Bax and active caspase 3. 116 Cryptotanshinone and dihydrotanshinone could prevent the growth of MDR-resistant SW620/Ad300 cells by inducing autophagic cell death and p53-independent cytotoxicity. 117 Hypericin, a natural anthraquinone, is a well-studied photosensitizer. Hypericin-mediated photodynamic therapy could resensitize CRC-resistant cells including HCT-8/L-OHP and HCT116/L-OHP cell lines toward L-OHP, which is associated with decreased drug efflux (downregulation of MRP2 level), GSH-related detoxification, and nucleotide excision repair (NER)mediated DNA repair. 118

| Others
Salvianolic acid B, a phenolic acid isolated from the dried root and rhizome of Salvia miltiorrhiza Bge. (Labiatae), could reverse MDR in HCT-8/VCR cells through elevating ROS levels, which promoted apoptosis and lowered the expression of P-gp, which increased the chemosensitivity of drug-resistant cancer cells. 122

| NEW DRUG DELIVERY SYSTEM
The low solubility and bioavailability, off-target, and cytotoxicity to normal tissues of chemotherapy decreases the therapeutic effect and promotes drug resistance in CRC, which causes relapse and poor prognosis of CRC patients. To deal with it, a new drug delivery system including nanocarriers, liposomes, exosomes, and hydrogels has been used to overcome chemoresistance in CRC.

| Nanocarriers
Lower toxicity and fewer side effects in blood and noncancerous tissues, increased aqueous solubility and bioavailability, specific target feature and multifunctional drug combination make nanoparticle-based formulations become an effective strategy to overcome drug resistance in CRC combined with conventional chemotherapy. 123 Chen et al reported that codelivery 5-FU with epidermal growth factor (EGF) grafted hollow mesoporous silica nanoparticles (EGF-HMSNs) can augment cytotoxicity and reverse MDR by enhancing drug accumulation in tumor cells, inducing S phase arrest and inducing cell death in 5-FU-resistant SW480 cells. 124 Using aptamer-conjugated grapefruit-derived nanovectors (GNVs) loaded with DOX and P-gp siRNA also showed MDR reversal effect, which could effectively inhibit proliferation and enhance apoptosis in MDR Lovo cells and the effect might be related to the downregulation of P-gp. 125 Other multifunctional codelivery systems, like mesoporous silica-coated gold nanorods (GNRs/mSiO2) loaded with DOX, conjugated with pH-responsive poly-histidine (PHIS), and covered with d-αtocopherol polyethylene glycol 1000 succinate (TPGS) was discovered to increase the drug accumulation and promote photothermal conversion in tumor resistant CRC cells and exert better anticancer effects in SW620/ Ad300 xenograft mice without observed systemic toxicity compared with other chemotherapy or photothermal therapy alone. 126 Combination use of sodium butyrate and Fe3O4 magnetic nanoparticles (MNPs), coated with folic acid (FA) and polyethylene glycol (PEG) (FA-PEG@ MNPs), could decrease cell viability and increase FA-PEG@MNPs intracellular uptake in Lovo cells, which indicated that the combined use may exert MDR resistance reversal effect in CRC. 127 Due to the multiple advantages of nanocarriers, recently, more studies are focusing on nanocarriers and trying to find different ways to overcome chemoresistance. 128,129

| Liposomes
Liposomes, artificial colloidal vesicles with unique lipid bilayered membranes, can target tumor cells after chemical modification or physical treatment and have been used in the targeted therapy of cancer. 129 Liposomes, as one kind of nanocarriers, own the advantages like biocompatibility and low systematic toxicity as well, which also contribute to drug resistance reversal. Xu et al found that bifunctional liposomes loaded with hypoxia-inducible factor-1(HIF-1) inhibitors, acriflavine (ACF), and DOX (DOX-ACF@Lipo) can reduce the chemoresistance to DOX in advanced CRC. 130 Combining 5-FU with a biochemical modulator in one same stealth liposome can exhibit significant antiproliferative activity and reverse drug resistance in resistant CRC cells. 131 Liposome encapsulation of VCR and monoclonal antibody or verapamil can effectively overcome MDR in human colon cancer cells. 132 Injectable PEGylated liposome encapsulating disulfiram was found to reverse chemoresistance at low nanomolar concentrations. 133

| Exosomes
Exosomes are endocytic extracellular vesicles (EVs) ranging from about 40-160 nm in diameter, which is associated with cancer proliferation, migration, invasion, and drug resistance. 134,135 Exomes induce drug resistance mainly by exporting chemical drugs from tumors or importing resistance-associated small molecules to cancer cells. CRC-derived exosomes have been found to enhance CRC chemoresistance by transferring cancer-related miR-NAs, 136 lncRNAs, 137 and circRNAs. 82,138 Due to their targeting specificity, high bioavailability, low immunogenicity, low toxicity, and good cell-cell communication capacity, exosomes have been employed as a new drug delivery system for drug resistance reversal in cancer treatment, including CRC. 139 Exosomes can overcome chemoresistance by transferring therapeutic ncRNAs and chemical drugs. It was reported that engineered exosomes containing miR-21i and 5-FU can effectively overcome drug resistance and dramatically elevated the cytotoxicity of 5-FU in the HCT-116/5-FU cell line, compared to the single use of either miR-21i or 5-FU. 34 Exosomal miR-204-5p sustained released by human-constructed HEK293T cells enhanced the 5-FU sensitivity of LoVo and HCT116 cell lines, and inhibited neoplasm growth in HCT116 xenograft mice. 140 Engineered exosomes loaded with lncRNA PGM5-AS1 and L-OHP realized by electroporation reversed L-OHP resistance in DLD1/L-OHP cell line and xenograft mice. 141 Exosomes-carried circ_0094343 improved the chemosensitivity to5-FU, L-OHP, and DOX via regulating the miR-766-5p/TRIM67 axis. 81 Circular RNA F-box and WD repeat domain containing 7 (circ-FBXW7) encapsulated in exosomes by electroporation could enhance the chemosensitivity of SW480/L-OHP and HCT116/L-OHP cells to L-OHP, increase apoptosis and inhibit EMT by binding to miR-18b-5p, the reversal effect was also found in the HCT116/L-OHP xenograft mice. 142 Kim et al. found that encapsulation of PTX to exosomes released from macrophages (exoPTX) by means of sonication could overcome MDR in resistant cancer cells by delivering the anticancer drug into the cancer cells directly, increasing drug accumulation and cytotoxicity, and bypassing the P-gp-mediated drug efflux. This effect was also verified in the murine model of lung metastases, exoPTX could significantly inhibit the tumor growth in the pulmonary. They also found that exosomes incorporated with doxorubicin (exoDOX) promoted the accumulation of DOX in resistant cancer cells. 143 After a modification of PTX-loaded exosomes with aminoethylanisamide-polyethylene glycol (AA-PEG) vector moiety, PTX exhibited more potent anticancer effect in lung metastasis compared to the single administration of exoPTX or PTX. 144

| Hydrogels
Hydrogels, an emerging drug carrier, are threedimensional (3D) frameworks comprised of cross-linked hydrophilic polymers. Due to their excellent biocompatibility and biodegradability, hydrogels have been extensively utilized for the treatment of cancer chemoresistance and relapse in recent years. 145 Hydrogels could modulate drug release when exposed to external stimuli including light, temperature, pH, and electrical and magnetic fields. 146 It has been found that hydrogel carriers loaded with drugs could overcome chemoresistance in many cancers, mainly including breast cancer, 147-149 ovarian cancer, 150,151 gastric cancer, 152 and CRC. 5-FU loaded in a thermal sensitive hydrogel system (a biodegradable PEG-polycaprolactone (PCL)-PEG (PECE) triblock copolymer), prolonged drug release time and markedly inhibited the growth and dissemination of CT26 cells in colorectal peritoneal carcinomatosis (CRPC) murine model. 153 An alginate nanocomposite hydrogel coloaded with CDDP and gold nanoparticles (AuNPs), combining photothermal therapy with chemotherapy, dramatically inhibited the growth of 90% colorectal tumors of control and dramatically improve the animal survival studied in CT26 colorectal xenograft murine model. This chemophotothermal therapy based on hydrogel could directly transport high-dose drugs and heat to tumors with neglectable side effects, which elevated the antitumor efficiency of CDDP and had the potential to evade CRC relapse. 154 A thermo-sensitive hydrogel coloaded with L-OHP and tannic acid (TA) polymeric nanoparticles (L-OHP/TA NPs), an injectable drug delivery system, could evade the growth of tumors and prolonged the survival time of the CT26 peritoneal CRC murine model. 155 An injectable thermal-sensitive poly(L-glutamate)-based hydrogel coloaded with CDDP and combretastatin A4 disodium phosphate (CA4P), exerted enhanced antitumor efficacy in C26 colon cancer-bearing mice. 156

| COMBINATION THERAPY
Combination therapy is the common treatment for CRC. FOLFOX, capecitabine and oxaliplatin (CapeOX), and leucovorin, fluorouracil, and irinotecan hydrochloride (FOLFIRI) are the first-line regimen for chemotherapy in CRC. Combination therapy could enhance anticancer efficacy, prolong survival, prevent drug resistance, and tumor relapse, which always makes an effect of "1 + 1 > 2." Various combination therapies combating chemoresistance have been clarified in the upper five sections, so the combined therapies not belonging to these sections are illustrated below.

| Combined with 5-FU
Auranofin, a forkhead box 3 (FoxO3) agonist and thioredoxin reductase 1 (TR1) inhibitor, could enhance the sensitivity of HCT-8/5-FU and SW620/5-FU cells to 5-FU in vitro and the sensitivity of SW620/5-FU cells to 5-FU treatment in vivo by inhibiting the nuclear factor erythroid 2-related factor 2 (Nrf2)/TR1 signaling pathway. Auranofin in combination with 5-FU induced the most cell death compared to treatment with auranofin or 5-FU alone and suppressed tumor cell invasion obviously. In SW620/5-FU cells xenograft nude mice, fewer lung metastatic nodules were observed in auranofin plus 5-FU group than in the 5-FU or auranofin groups. 157 It has been found that Lactobacillus Plantarum Supernatant (LP SN) combined with 5-FU could reverse the resistance to 5-FU and enhance the therapeutic effect of 5-FU in 5-FU-resistant HT29 cells and HCT116 cells by inactivating the Wnt/β-catenin signaling, reducing CSCs, and increasing apoptosis. 158 Curaxins, a class of small molecules, might overcome 5-FU resistance in defective mismatchrepair (dMMR) colorectal cancer patients by targeting the histone chaperone facilitates chromatin transcription (FACT) complex. 159

| Combined with L-OHP
Administration of Toll-like receptor agonists R848 combined with L-OHP reversed the functional orientation of myeloid-derived suppressor cells (MDSCs) toward tumoricidal M1-like macrophages and strengthened the antitumor effect of L-OHP. 160 Combinational inhibition of YAP by a YAP-specific inhibitor verteporfin and an inhibitor of EGFR/ErbB1 AG1478 synergistically inhibited the CRC progression and overcame chemoresistance. 161 GW4064, a synthetic farnesoid X receptor (FXR) agonist, could enhance the antineoplasm effects of L-OHP by inducing BAX/caspase-3/ gasdermin E (GSDME)-mediated pyroptosis in HT26 and SW620 cells. 162

| Combined with DOX
Tamoxifen, a selective estrogen receptor modulator, combined with DOX can reverse MDR in CRC nude mice, reduced weight and volume of transplanted tumors, which is independent of the expression of estrogen receptors and has no influence on MDR1 expression. 163

| CONCLUSION
CRC is one of the most prevalent cancers across the world with increasing incidence in recent years, and the chemoresistance caused by long-term use has evaded the therapeutic process of CRC. Chemical drugs, combined with clinical common drugs, herbal medicine monomers, gene therapy, and protein inhibitors, could restore the chemosensitivity of tumors to chemodrugs. Nanocarriers, liposomes, exosomes, and hydrogels, as drug delivery systems, could combine multiple drugs together well, transport these drugs to the tumor site directly, and control the drug release, which dramatically enhances the drug effect and reverse the drug resistance occurred in monodrug therapy. Reversal drugs involved in drug repurposing and herbal medicinal compound are already marketed drugs, and are the safest and fastest mean able to apply in the clinic, thus more efforts should be contributed in these fields. Gene therapy and protein inhibitors, belonging to gene and protein levels, respectively, become a hot spot in cancer treatment and chemoresistance. Moreover, gene therapy in chemoresistance reversal still stays in the early stage but own huge developmental potential. So far, although various potential strategies have been found, there is still a long way to go from laboratory to clinic. Therefore, further and consistent investigation is required to the realization of the victory in the chemoresistance combat of CRC.