The most common cancer in 2019 was breast cancer, followed by lung and prostate cancer. 1 There are different types of cancer treatment including conventional single treatment or a combination of other treatments. The primary therapy includes surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, or precision medicine. 2 The three main agents widely used to alter cancer development through signaling pathways are small molecules, nucleic acids and antibodies. However, small molecule inhibitors are widely studied but have drawbacks in terms of solubility, bioavailability, cellular targeting, and toxicity in normal cells. 3 Most treatments result in high morbidity and reduced quality of life. Researchers are focusing on existing drugs and using them for cancer therapy because of their known profiles and high success rates. For the reuse of age-old used drugs, the clinical approval time is also shorter and the success rates are significant. 4 Small molecules face different challenges when acting in different pathways, particularly due to their complexity within the pathway. For example, Wnt (Wingless-related integration site), Notch (Neurogenic locus notch homolog protein) 1 , and Hedgehog signaling pathways are interconnected and crossed downstream. These genes acts as a tumor promoter in one pathway and as a tumor suppressor in another. 3 Much research has been conducted on the safety of quinacrine since US soldiers widely used it; About three million people were treated during World War II. For the identification of anticancer drugs, preference has been given to QC because it has low toxicity in normal cells and has no ff-target effects that might limit chemotherapy. 4 1920s. 5 QC is commonly used as mepacrine, quinacrine hydrochloride, quinacrine dihydrochloride and atabrine (registered name) to treat diseases other than malaria, giardiasis, tapeworm infection, refractory lupus erythematosus, rheumatoid arthritis and even as adjuvant cancer therapy. QC was rediscovered in a blind chemical library screen for small molecules that can activate p53 in tumor cells without causing genotoxicity. There is a need for a rationally designed drug that can act like a shotgun and be targeted in different or multiple pathways. 6 Its effects on several important signaling pathways involved in the malignant progression of numerous cancers make QC an exciting candidate as a chemotherapeutic agent for novel combination treatments. 7 QC has been shown to be a chemosensitizer that can enhance the chemotherapeutic effect on cancer cells, opening a new dimension for combination therapy. 8

WNT signaling can be broadly categorized as canonical and non-canonical pathways, with canonical or B-catenin pathways playing critical roles in cancer progression. Much work has been done in recent years to provide a detailed insight into the function and regulation of each stage of the canonical path, which has proved complicated. 9 APC and β-catenin play critical roles in proliferation, differentiation, and migration associated with multiple proteins in signaling pathways involved in attachment and cell cycle signaling. 10 Research shows that QC exposed breast cancer cells activate apoptosis by APC in the WNT-TCF pathway. Upregulation of APC with significant downregulation of GSK-3 (Glycogen synthase kinase-3) and Axin has been noted. APC, a multidomain protein, forms an Armadillo repeat domain, 15 residues, 20 residue domains and SAMP repeats, and a primary domain of which 15 amino acid repeats and 20 amino acid repeats are residues. This is essential to negatively regulate canonical signaling by binding to CTBP1 and CTBP2. Both of these transcriptional co-repressors block β-catenin and stimulate APC oligomerization. 11 APC also regulates microtubule stabilization, kinetochore function, and chromosomal segregation through basic and C-terminal domains (Zhang and Shay 2017), thereby inhibiting PARP and disassembling the BER complex. In the presence of QCABT 888, a PARP inhibitor has also shown increased DNA damage-inducing apoptosis in breast cancer cell lines. 12 An increase in cytotoxicity leading to apoptosis has been identified through DNA damage, cell cycle arrest and topoisomerase activity via the WNT-Tcf pathway. 13

Hedgehog signaling can be activated classically or non-classically, both of which require the involvement of GLI. Although the end result of this pathway is proliferation and differentiation, different cancer type activation pathways are observed to alter their mode of action. 14 Since metastasis and drug resistance are the major barriers to curing cancer, most drugs are observed to have the potential to stop cancer metastasis by blocking the hedgehog GLI axis. 13 SMO, a GPCR-like protein, is activated by PTCH1 and translocated to the ciliary membrane after launch, which is essential for GLI activation. GLI, an oncogene activation, has also been shown to induce the modulation of Wnt signaling. 14 Recently, it was confirmed that QC significantly reduces the expression of SMO in triple-negative breast cancer while there is no significant toxic effect on normal breast cells. 15 A 2016 study proved that NQC (Nano Quinacrine) is more potent and has the potential to induce cytotoxicity and, therefore, apoptosis than QC. Recent research has showed that there was downregulation of GLI, c-Myc, and Cyclin D and no changes in the sub-G1 population. GSK-3, a negative regulator of HH-GLI, has shown upregulation of protein expression after NQC exposure. Because QC is an intercalating agent, it inserts itself between the GLI-DNA bond, thus destabilizing the complex. The GLI-DNA complex is an essential phenomenon for downstream activation and GLI-dependent cell survival. NQC binds between two GC base pairs and thus damages DNA. 13

Notch pathways play important roles in cancer metabolism, cell survival and also maintenance of cancer stem cells from cancer. It is also through this pathway cancer cells shifts from epithelial to mesenchymal transition to mesenchymal to epithelial cells and viceversa 16 . The Notch pathway is mostly considered non-functional in cancer, but it also depends on the type of cancer. It could serve as a tumor suppressor, while it has been shown to be oncogenic when upregulated. 3 Kundu et al. have mentioned that no commendable changes in the expression of cleaved NOTCH 1, an activator of the NOTCH cascade, were observed. 13 In 2019, they observed that NQC exposure induced nuclear translocation of nectin-4 and downregulated ADAM-17 expression. In combination treatment with 5-FU and NQC, the expression of nectin 4 was downregulated and there were no other changes in the NOTCH pathway. 17 Musashi proteins are RNA-binding proteins encoded by MSI1 and MSI2 that regulate transcription events and cellular regulation. These relate to the prognosis of glioma, brain tumor, breast cancer and colon cancer. In contrast, when overexpressed, Numb inhibits cell proliferation, migration, and invasion due to downregulation of cyclin D1 or MMP-9 expression. 1819 QC has been shown to downregulate MSI2, induce expression of Numb, which inhibits cell cycle progression at G0/G1 phase, repress c-Myc, and downregulate CDK4 and CDK6 expression. This study demonstrated that QC mode of action in DLBCL (diffuse large B cell lymphoma) is heterogeneous and mediates through the MSI2-NUMB pathway. 18

The JNK signaling pathway includes an important MAPK signaling pathway that controls proliferation, migration, development, and apoptosis. There are 13 different MAPK signals that can be activated by the JNK pathway, each inducing a different cellular function, metabolism, inflammation, and cytokine production. Once started, this pathway can lead to apoptosis by two different mechanisms. The c-Jun/Ap1-dependent pathway involves BAX/BCL2, cytochrome-c and Apaf-1, and the other involves Smac/Diablo through caspase8 activation. 20 Quinacrine induces ROS generation in various cancers, ultimately leading to apoptosis. ROS has been shown to phosphorylate the MAPK pathway QC does not alter JNK phosphorylation in K562 cells although phosphorus-ERK is reduced. QC acts by inducing p38 phosphorylation and inactivating ERK, independently. Apoptosis is also activated by repression of BCL2, BCL2L1 and translocation of BAX in the nucleus when QC is exposed, which depends on p38 and p-ERK regulation. Reducing ERK has proved to involve HSP70 folding and mitigating cancer promotion. 91 BCL2 is regulated by AP1, which consists of homodimers and heterodimers of c-Jun, c-Fos, and ATF-2. QC induces the ERK pathway by downregulating phospho-c-Jun and phospho-c-Fos without altering ATF-2 and MCL1 expression. All of these steps cumulatively lead to apoptosis by releasing cytochrome c and activating caspase 9 and caspase 3. 21 Furthermore, the elimination of Mcl-1 has been shown to be a critical point in inducing apoptosis, and in the presence of a low dose of QC, down-regulation of MCL1 is promising. 22 Janie et al. demonstrated that BCL2 can also be induced by the QC-activated NF-k pathway in colon cancer cells 23 , indicating that apoptosis induction by QC is cancer-and cell-origin dependent.

JNK regulates various cellular processes including proliferation, differentiation, apoptosis, and DNA repair, and its activity depends on GSTA1 expression. GST upregulation, arrest of apoptosis, abnormal cell growth and the JNK signaling pathway are observed to be linked and interdependent. 24 QC inhibits GSTA1 by binding to the G site and tyrosine 9 through hydrogen bonding, which is the catalytic subunit of GSTA1. An increase in ROS generation due to inhibition of GSTs has been studied, resulting in blockage of phosphorylation of eIF2a. 25 PLA2 has a significant association with prognosis in multiple tumors by modulating signaling pathways including proliferation, tumorigenesis and metastasis. 26 The cytosolic PLA2 enzyme has been shown to be activated and phosphorylated by MAP kinase. QC inhibits the action of PLA2 by downregulating it. 27

EGFR, a proto-oncogene, plays an important role in cell proliferation and cell survival. Small aberrations in this gene can cause changes in cyclin/CDK 4-6, causing oncogenesis. 28 EGFR activation involves a number of activations of different signaling pathways, including P13K, AKT, ERK and signaling pathways leading to apoptosis and migration. 29 KRAS mutant lung cancer cells suppress autophagy upon exposure to QC, while inhibition of autophagy does not induce apoptosis in KRAS mutant cells. Additionally, inhibition of oxPPP (an oxidative arm of the pentose phosphate pathway) has been shown to promote autophagy inhibition while increasing cytotoxicity in cells. This study focuses primarily on PDAC and NSCLC cancer types, most of which have KRAS mutant cells. Nevertheless, QC has been found to block autophagy and oxPPP and stimulate cytotoxicity and hence cell death regardless of status. 30 FER tyrosine kinase is a non-receptor tyrosine kinase that is activated downstream of EGFR, regulates cell adhesion and sends signals from the cell surface to the cytoskeleton. It is observed to be overexpressed in malignant cells compared to its standard counterpart, but its relevance depends on the type of cell and the stimulus. 3132 Once overexpressed, FER increases phosphorylation of EGFR, ERK, and NF-k in the presence of QC EGFR binds directly to the SH2 domain of FER with phosphotyrosine residues of EGFR, thereby activating ERK and NF-k signaling pathways. QC exposed cells are more effective in the average number of EGFR and FER expressed cells. While overexpression of these proteins will terminate the effects of QC, resulting in better potency, the ERK inhibitor will support exposure. 32

The Rho signaling pathway plays a crucial role in cancer progression in (i) cell cycle, (ii) morphogenesis and (iii) migration. 33 In our laboratory, we observed different types of modulations of the cytoskeleton, such as B. lamellipodial protrusions and the formation of filopodial structures leading to the expression of Rac1, RhoA and Cdc42. Our study sheds light on the activation of Rac1 and Cdc42 after QC exposure to show a decrease in RhoA expression. QC was able to downregulate the master gene for modulation of the cell cytoskeleton, which is RhoA, since RhoA plays a role in tumor cell proliferation, cell survival and stimulation of transformation. 34 During motility, previous investigators observed that Rac activation at the front and Rho activation at the back promote migration. 3335 The lack of migratory ability with QC concentration could also be attributed to the downregulation of RhoA.

The AKT pathway is an essential pathway related to the stimulation of growth factors and other cell functions such as cell growth, apoptosis, and cell survival. Most studies have proven that tumor aggressiveness is the result of the malfunction of the AKT pathway and its proteins namely Eif4E, periostin, both the p110 and p85 subunits of PI3K. The mTOR-activated pathway negatively regulates autophagy, making it an unavoidable target for targeting PI3K/Akt/mTOR pathways and thus reducing angiogenesis. 37 9AA or QC blocks AKT phosphorylation at Ser473 and thus inhibits AKT and mTOR function, since mTOR is the essential component of TORC2 that phosphorylates Ser473. 36 mTOR is also a component of mTORC1, which has its effect on 5cap-dependent Mrna translation by 4EBP1. mTOR phosphorylates 4EBP1 and releases Eif4E, which initiates translation. 37 QC inhibits mTOR and mTOR-mediated translation, which in turn downregulates p110 protein. Since p110 is the class IB catalytic subunit of the P13K family, the pathway is also blocked by QC exposure. P13K induction is required for phosphorylation and activation of the AKT pathway, which sequentially activates THE NF-k pathway by phosphorylation of p65 and Mdm2 to start the p53 pathway. 36 When a hybrid of the QC and the 13thiazinan-4-one group was prepared, the anticancer effects were enhanced. The hybrid affected mTOR-4E-BP1 directly without downregulating the AKT pathway, unlike QC. The compound is also successful in downregulating all genes affecting the mTOR pathway downstream. 38

Cell metabolism and reprogramming are critical to cell sustenance, growth, and proliferation. This cellular metabolism is the connecting link between cell signaling and maintains the microenvironment and thus critical points of cancer, which is mainly influenced by p53. 39 QC increased p21 and MDM2 protein levels and also induced stabilization and accumulation of p53 protein. In addition to acting as a DNA damaging agent, QC also activates p53 and its transcription which is dose and time dependent, and also restores the wild-type p53 in UM-SCC-HNSCC cell lines by altering the TP53 mRNA and protein expression induced. 4 9AA was found to be less apoptotic in renal cell carcinoma in the presence of null p53. It is noted that the QC mode of anticancer action and dependence on p53 makes it different from traditional anticancer agents, e.g. B. camptothecin, doxorubicin, taxol and vinblastine. QC does not phosphorylate p53, but Ser-392 of the protein kinase CK2. 40 P53 plays a critical role in tumor cell susceptibility to TRAIL, particularly when TRAIL is combined with DNA-damaging chemotherapy or radiation to treat wild-type p53-expressing tumors. QC has been shown to enhance TRAIL-DR5 interaction, which ultimately increases cellular apoptosis. 2341 When QC and TRAIL were administered, ROS production, BAX and caspase activation increased synergistically, leading to apoptosis by damaging mitochondria through the intrinsic pathway of apoptosis. 4243 QC enhances the binding affinity of TRAIL-DR5, resulting in TRAIL binding to the ectodomain of DR5. Subsequently, FADD is recruited, which recruits the procaspase 8 for the activation of caspase8. FADD and caspase8 together form DISC, the death-triggering complex, hence apoptosis. 42 QC has been shown to increase the half-life of DR5 in the ovarian cancer cell line, leading to the accumulation of DR5 and altering its subcellular localization in lysosomes. 44 The main regulatory factor that controls cell survival and apoptosis occurs through cell cycle regulation, mainly through one of the phases G0/G1, S or G2/M. QC has been shown to enrich cells in the S phase of the cell cycle and thus inhibit proliferation in the gastric cancer cell line. P53 was strongly upregulated upon drug exposure, confirming apoptosis. In the gastric cancer cell line, QC upregulated Bax and downregulated Bcl-2, releasing cytochrome c and activating caspase 3, resulting in the intrinsic pathway of apoptosis. 45 Cell death can be divided into three types: apoptosis, autophagy and necrosis. Many anticancer drugs induce cell death through autophagy other than apoptosis, suggesting that it plays a critical role in tumor cell growth and differentiation. 46 First and foremost, QC, an anticancer agent, has been shown to act in the late stages of autophagy inhibitor. 30 It was later found to stimulate autophagy in various cancer cell lines. The mode of action turns out to be an excessive accumulation of autophagic vacuoles and their inability to degrade vacuoles and an increased expression of LCB3. Weak base QC traps itself in the lysosome, leading to increased pH and thus cell death via the p53 and p21 pathways. 47 One study confirmed that there is a dramatic increase in LC3-II expression upon exposure to QC and hypothesized that QC drives cells to autophagy, increasing stress within cells and thus inducing apoptosis. 4 In the breast cancer cell line, QC caused apoptosis by upregulating expression of both p53 and p21 genes and inhibiting topoisomerase activity. QC supercoils the DNA, resulting in the inhibition of replication; therefore, cells in S phase aggregate to cell death 48. This cessation of apoptosis or autophagy is decided by exposure to QC, and TRAIL is decided by activating two processes, p21 or DR5, in different breast cancer cell lines. Upregulation of these key proteins is the main feature of QC and TRAIL's synergistic effect with downregulation of other pathway proteins mTOR/P13K/AKT. 49 Hsp70 prevents cell death by interfering with the ability of cytochrome c and Apaf-1 to recruit procaspase9. Hsp70 can block the release and activation of procaspase 9, which is functional for cell survival relative to cell death. Our study implies that QC increases Hsp70 downregulating Caspase9 expressions. It is evident that Hsp70, the antiapoptotic gene, probably blocks expression of the apoptosome by caspase9 and therefore does not lead to apoptosis via the intrinsic pathway. 34 Colon cancer cells normally undergo the intrinsic pathway of apoptosis in the presence of QC, but as the cytotoxicity of the cells increases (QC dose increases), the cells tend to reach apoptosis via the extrinsic pathway of apoptosis. 3450 QC EXPOSURE INHIBITS NF-kβ SIGNALING PATHWAY NF-k, a family of five transcription factors that regulate cellular processes, plays important roles in cancer initiation, development, metastasis, inflammatory microenvironmental activity, and exceptional resistance to cancer treatments. This pathway is activated by multiple stimuli, namely cytokines, growth factors, bacterial, viral products, UV or ionizing radiation, ROS, DNA damage, and oncogenic stress. 51 QC has been shown to induce p53 and inhibit NF-kβ in renal cell carcinoma. In highly expressed NF-kβ cell lines, they are sensitive to QC, ignore p53 status and vice versa. Once activated, NFkβ induces TRAIL and L-OHP in the cell lines mentioned; decreases c-FLIP and Mcl-1 without affecting NF-kβ dependent proteins. NF-kβ, in the presence of QC, binds to the promoter region of c-FLIP and MCL-1 and shortens their half-life, demonstrating that these two genes are key factors in NF-kβ regulation. 23 The suppression of NF-kβ by TNF induction proved to be another function of this pathway. QC induced nuclear translocation, accumulation of NF-kβ and also increased its presence in nuclei. 40 Heat shock response signaling is important for tumor survival and pyogenesis; QC represses Hsp70 synthesis by reducing HSF-1 dependent transcription. 8 Figure 2. Graphical representation of the mode of action of Quinacrine. QC upregulates p53 and leads to caspasemediated apoptosis. HSP70 was upregulated, resulting in a stress response and thus cell death. At a higher time point of QC exposure, the HSP70 fold was blocked by P-ERK downregulation. Once P-ERK is downregulated, the classic properties of cancer cells are disrupted, epithelial-to-mesenchymal transition (EMT), migratory ability, and invasiveness are disrupted. QC directly affects cytoskeletal modulations by acting on small GTPases, namely Rac1, RhoA and Cdc42. ROS production increases due to QC exposure and various stress responses, leading to cell death through apoptosis.

BMPs (Bone Morphogenesis Protein) are multifunctional cytokines belonging to the TGF family that modulate tumor growth, differentiation, and apoptosis in various cancers. Depending on the type of cancer tissue and its epigenetics, BMPs can act as tumor suppressors or promoters. They have been shown to activate numerous essential proteins downstream, such as SMAD, which regulate p38, JNK, Akt, LIMK, Rho, Rock proteins. 52 QC has been shown to induce the expression of the BMP signaling genes BMP-2 and BMP-4, four times higher than average, and upregulates TGF-1 to induce TGF-β. 55 Protein is associated with tumorigenesis, and drug resistance and thus a crucial target for cancer therapeutics 53

Protein takes an active part in tumor cell survival, cell proliferation, and drug resistance 54

Regulates multiple cellular processes that can promote tumorigenesis, including proliferation, migration, invasion, and angiogenesis 76 SOX2

An anticancer target promoting proliferation, survival, invasion/metastasis, cancer stemness, and drug resistance 55

It regulates cell migration, invasion, and anoikis resistance in breast cancer cells 56 small-GTPases

The Raf-MEK-ERK pathway is a crucial downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers 57

A frequently modified gene in all types of human cancer, playing key roles in the actin-microtubule cytoskeleton and gene transcription 58 AKT SIGNALING PATHWAY mTOR Proved to have interesting mechanisms as a potential benefit for developing anticancer drugs 59

Has pro-tumorigenic activity and is mainly found inactive in different types of cancer 60 p53 SIGNALING PATHWAY p21 A tumor suppressor gene stimulated in the presence and independent of p53 82 MDM2

An attractive treatment for cancers and wild-type p53 61

A stimulator of apoptosis and diverse intracellular signaling pathway in cancer 62

The ratio profoundly defines the resistance or submission to apoptosis in cancer 63

It helps the proliferation of cancer by suppressing multiple apoptotic pathways, regulating necrosis, bypassing cellular senescence program, interfering with tumor immunity, promoting angiogenesis, and supporting metastasis 64 NF-kβ SIGNALING PATHWAY TNF An inflammatory cytokine having elevated expression in a variety of cancer cells 65

Dominates critical roles in cancer progression like inflammation, cancer cell proliferation, and survival, EMT, invasive behavior, angiogenesis and metastasis, genetic and epigenetic alterations, and cancer stem cell formation 88 TGF-β SIGNALING PATHWAY BMP-2 Elevated levels of BMP2 promote liver cancer 66

Expression levels are commonly altered in tumors, and it is linked to patient prognosis 90

Currently, there is a need for a rationally designed drug that can act as a shotgun and target different or several pathways. 6 QC affects multiple keys signaling pathways, implicated in the malignant progression of numerous cancer types, making it an exciting candidate as a chemotherapeutic agent for new kinds of combination treatments 7 . QC has been proved to be a chemosensitizer that can enhance chemotherapeutic effects on cancer cells, thus opening a new dimension for combinational therapy. The antitumor property was observed in both in vitro as well as in vivo studies. 50 QC's anticancer properties are well established in many tumor cells. 6768 A further report confirmed that QC could activate p53 without genotoxicity through protein stabilization by blocking p53 ubiquitination. This deregulation results from the phosphoinositol-3 kinase/AKT/mammalian target of the rapamycin pathway. 34 QC binds and inhibits proteins involved in multidrug resistance, disrupting the arachidonic acid pathway and affecting the p53, NF-ĸβ, and AKT pathways. 6 QC offers anticancer potential by downregulating cellular inhibitors of apoptosis protein-1, upregulation of Bax, and cleaving caspase 3 independent of p53. QC activates p53, a transcription factor for cell cycle arrest, cell proliferation control, DNA repair, and apoptosis. 69 Therefore, Quinacrine has the potential to be used as one of the small molecules that could be repurposed for cancer chemotherapeutic management in the near future.