PTOX, whose pharmacological effects have been recognized to inhibit microtubule assembly by blocking the colchicine binding site, thereby exhibiting inhibition of microtubule protein polymerization and inducing G2/M blockade, has shown potent antitumor activity (Cortese et al., 1977). PTOX derivatives have also been shown to have the analogous ability (Han et al., 2018). Kamal et al. (2011b) synthesized a series of conjugates of 4-aza-2,3-didehydropodophyllotoxins Compound 21 (Figure 7), and fluorescent tubulin polymerization analysis showed these PTOX derivatives significantly reduced tubulin units. On the molecular mechanism, these PTOX-based conjugates caused apoptosis of A549 cancer cells in a caspase-3 dependent manner (Kamal et al., 2014). Likewise, Kandil et al. (2016) synthesized 4-azapodophyllotoxin derivatives Compound 22 (Figure 7), which induces disruption of the microtubule cytoskeleton at a sub-micromolar level to MCF-7 cancer cells. Similarly, 4β-[(5-substituted)-1,2,3,4-tetrazolyl] PTOX derivatives Compound 23 (Figure 7) were synthesized, which inhibited nearly 90% polymerization of tubulin at only 5 μM concentration (Hyder et al., 2015). Sadeghi et al. (2015) investigated the antitumor effect of 6-methoxyPTOX (MPTOX) Compound 24 (Figure 7), which has a similar structure to PTOX. Treatment of human bladder cancer (5637) cell line and myeloid leukemia (K562) cell line with MPTOX significantly reduced the viability of tumor cells and induced their programmed cell death. In addition, the expression of TUBB3 (a key member of β-tubulin) and TOPIIA (a key nuclease in DNA replication) was inhibited in tumor cells after MPTOX treatment. Furthermore, Bai et al. (2012) and Zhao et al. (2015, 2019) demonstrated that the carbon-sulfur bond at the 4-position (C-4) of the carbon ring of PTOX reduced the inhibitory effect of dose on tubulin polymerization, thereby enhancing its therapeutic effect. They synthesized 4β-NH-(6-aminoindole)-4-deoxy-PTOX Compound 25 (Figure 7) that can target the α-tubulin binding site and colchicine binding domain, as confirmed by X-ray crystallographic analysis. Importantly, these PTOX derivatives exhibited nanomolar antitumor efficacy in vitro and no significant toxicity in vivo.

The severe side effects of PTOX, such as high toxicity and gastrointestinal upset, have limited its use in cancer chemotherapy. Extensive structural modifications of PTOX have yielded more effective and less toxic anticancer agents, such as etoposide and teniposide, which have been widely used in cancer chemotherapy (Evans et al., 1985; Paz-Ares et al., 2019). They stabilize the cleavage complex formed between the enzyme and its DNA substrate, thereby inhibiting topoisomerase II degradation and ultimately triggering cell death through the accumulation of chromosome breaks. Topoisomerases (topo-I and topo-II) play an important role in DNA replication, transcription and are a group of promising antitumor targets. Scholars generally believe that the bulky motif at the C-4 position of PTOX may be responsible for the inhibition of topo II (Stähelin and von Wartburg, 1991), and γ-H2AX is a classical marker of double-stranded DNA breaks during DNA damage (Kinner et al., 2008). Kamal et al. (2011a) developed the new PTOX analog 4β-acrylamidopodophyllotoxin congeners Compound 26 (Figure 8) as a potential anticancer agent. Their results showed that a large number of γ-H2AX foci were observed in etoposide and this PTOX derivative-treated cells, while double-stranded DNA breaks were negligible in PTOX-treated cells. Shankaraiah et al. (2015) developed a facile one-pot method and successfully synthesized novel PTOX-thiourea hybrids Compound 27 (Figure 8), which showed selective DNA topoisomerase II inhibitory activity and anticancer activity against human prostate cancer (DU-145) cell line. In addition, the 4bβ-aminotriazole PTOX derivative Compound 28 (Figure 8) synthesized by Reddy et al. (2018) showed potent cytotoxicity on prostate cancer cell lines and was efficient in inhibiting DNA topoisomerase II. Likewise, β-carboline PTOX congeners Compound 29 (Figure 8) exhibited excellent cytotoxicity against human prostate cancer (DU-145) cell lines, and the DNA topoisomerase II inhibitory ability of these congeners was confirmed by comet analysis, DNA binding studies, and docking studies (Sathish et al., 2018).

In malignant chemotherapy, resistance to anticancer drugs can often be demonstrated and is usually manifested as multidrug resistance (MDR), which is one of the research hotspots in cancer prevention and treatment (Erin et al., 2020). The superfamily of transmembrane ATP-binding cassette (ABC) transporter proteins enhances the efflux of a wide range of chemotherapeutic agents and is regarded as one of the major causes of MDR in cancer, with the role of the highly expressed P-glycoprotein (P-gp) being the most well established (Zhang et al., 2021). Chen et al. (2009) synthesized a PTOX derivative, L1EPO Compound 30 (Figure 9), which exhibited cytotoxicity to P-gp-mediated MDR cancer cell lines (K562/A02 and KBv200) but was less cytotoxic to normal human cell lines. Furthermore, L1EPO downregulated the mdr-1 gene and reduced the expression of P-gp, which may be the mechanism of its anti-MDR effect. Similarly, YB-1EPN Compound 31 (Figure 9) as a PTOX derivative also showed cytotoxicity against KBV200 and K562/A02 cells, while downregulating mdr-1 and bcl-2 expression, suggesting its ability to overcome P-gp-mediated MDR (Chen et al., 2010). Hu et al. (2010) synthesized a range of 4β-anilino-PTOX analogs Compound 32 (Figure 9) that led to G2/M phase arrest and eventually induced apoptosis in drug-resistant KB/VCR cancer cells. In both drug-sensitive xenograft models and drug-resistant xenograft models, the administration of 4β-anilino-PTOX analogs can effectively treat cancer at lower doses than VP-16. In many other studies, it has been shown that 4β-anilino-PTOX analogs may kill MDR tumor cells (e.g., K562/A02) by downregulating mdr-1 expression (Cheng et al., 2014; Cao et al., 2015, 2018). Zhang et al. (2016a) synthesized pyridine acid esters of PTOX Compound 33 (Figure 9), which efficiently triggered cell cycle arrest and accompanied induction of apoptosis in K562/ADR tumor cells [resistant to adriamycin (ADR)]. Regarding the molecular mechanism, the results suggested that the pyridylate of PTOX may reduce the expression of the P-gp protein by stimulating the ERK1/2 signaling patway. In addition, aromatic heterocyclic esters of PTOX also showed potent anti-MDR activity against K562/ADR cancer cells, causing cellular S-phase block, apoptosis, and downregulation of P-gp expression, with molecular mechanisms suggesting an association with the ROS/MAPK pathway (Zhang et al., 2016b).

In addition, nanomedicine, which has been widely used in anti-cancer, can be used to modify PTOX derivatives to improve their poor solubility and high toxicity. Roy et al. (2015, 2017) developed PTOX-based nanomedicines by covalently coupling PTOX and polyethylene glycol (PEG) with acetylated carboxymethylcellulose (CMC-Ac) Compound 34 (Figure 9) via one-pot esterification chemistry. They prepared PTOX-based nanoparticles (NPs) with an average diameter of 20 nm, which released PTOX at ∼5%/day in serum. The NPs exhibited excellent killing ability and high selectivity for tumor cells, their accumulation in tumor tissues was eightfold higher than all other tissues, and they showed significantly improved efficacy against MDR tumors in mice with minimal systemic toxicity. Zhou et al. (2018) developed a peptide-based PTOX conjugated poly(_L-glutamic acid)-g-methoxy poly(ethylene glycol) (PLG-g-mPEG) Compound 35 (Figure 9), which effectively inhibited the expression of P-gp in the MCF-7/ADR cancer cells. In vivo experiments showed that the PLG-g-mPEG-PTOX conjugate had excellent antitumor efficacy against MCF-7/ADR xenograft tumors with a tumor inhibition rate of 82.5%. Compared with free PTOX, it showed significantly higher anticancer efficacy with minimal toxicity. In addition, Feng et al. (2020) developed a pH/redox cascade-sensitive multiscale nanoparticle, PAMAM-ss-PPT, which can fully release PTOX in the presence of elevated glutathione (GSH). Results demonstrated that nanoparticles can effectively penetrate paclitaxel-resistant A549 cancer cell xenografts and inhibit tumor growth with negligible toxicity (Feng et al., 2020).

In the studies on the antitumor function of PTOX derivatives, it was found that they can cause apoptosis and affect numerous related signaling pathways. Caspase-mediated activation is the canonical pathway for the induction of apoptosis. Precursor caspases will be proteolyzed upon receiving the apoptotic signal, and caspases-3, -8, and -9 are the most common apoptotic caspases among them (Porter and Jänicke, 1999; Kim et al., 2015; Tummers and Green, 2017). We thoroughly summarize the effects of PTOX derivatives on different cystatin caspases in cancer cells in the following (Table 2). (i) caspase-3: DPMA (Sang et al., 2013), 4-aza-2,3-didehydropodophyllotoxins (Kamal et al., 2011b, 2014), triazolo linked PTOX conjugates Compound 36 (Figure 10; Vishnuvardhan et al., 2017), deoxypodophyllotoxin (DPT) (Hui et al., 2016), biotinylated PTOX derivatives Compound 37 (Figure 10; Zi et al., 2019); (ii) caspases-8: hybrids of PTOX and formononetin (Yang et al., 2019); (iii) caspases-9: 4β-amidopodophyllotoxins (Kamal et al., 2013); (iv) caspases−3 and −9: OAMDP Compound 38 (Figure 10; Ren et al., 2018) and spin-labeled PTOX derivatives Compound 39 (Figure 10; Yang et al., 2017); (v) caspases−3, −8, and −9: β-apopicropodophyllin (Kim et al., 2018), PTOX acetate (Choi et al., 2015a,b; Hong et al., 2016), aromatic heterocyclic esters of PTOX (Zhang et al., 2016b), acid-PTOX conjugate Compound 40 (Figure 10; Zhang et al., 2017b); (vi) multiple caspases: picropodophyllotoxin Compound 41 (Figure 10; Kwak et al., 2020). In addition, PTOX may affect other signaling pathways to trigger cell apoptosis. Han et al. (2016) synthesized PTOX-norcantharidin hybrids Compound 42 (Figure 10), which showed low cytotoxicity against normal human embryonic kidney cells (293T) and induced cell cycle G2/M arrest and apoptosis in MCF-7 cancer cells. In terms of molecular mechanisms, the results showed an upregulation of the expression of the cell cycle-related protein CDK1 and downregulation of the expression of cyclin B1, a protein required for mitotic initiation. Wang J. et al. (2018) synthesized PTOX-indirubin hybrids Compound 43 (Figure 10), which resulted in apoptosis and cell cycle arrest in human leukemia cells (K562/VCR). Meanwhile, PTOX-indirubin hybrids elicited the accumulation of intracellular ROS, regulated JNK and AKT signaling, and downregulated the expression levels of P-gp and MRP1 proteins. On the other hand, it was revealed by Western blot that the hybrid could induce autophagy in K562/VCR cells by increasing the levels of Beclin1 and LC3-II.

Epithelial-mesenchymal transition (EMT) is a cellular program that is known to be essential for the development of malignant tumors. EMT can confer greater initiation and metastatic potential to cancer cells, as well as greater resistance to therapeutic modalities such as chemotherapy (Dongre and Weinberg, 2019). The natural PTOX analog 4′-demethyl-deoxypodophyllotoxin glucoside (4DPG), which increased the expression of checkpoint kinase 2 (Chk2), a tumor suppressor of the DNA damage checkpoint pathway. Overexpression of Chk2 eliminated the metastatic ability of tumor cells and reduced the expression of proteins associated with EMT (including Twist1, Snail1, and MMP-2) (Nayak et al., 2017). Likewise, 4DPG synthesized by Katoch et al. (2021) also enhanced the level of Chk2 and attenuated the expression of EMT-related proteins in human colorectal cancer (HCT-116 and SW-620) cell lines. Li et al. (2019b; 2019c) synthesized 2-pyridinealdehyde hydrazone dithiocarbamate S-propionate PTOX ester (Ptox^Pdp) Compound 44 (Figure 10) and di-2-pyridineketone hydrazone dithiocarbamate S-propionate podophyllotoxin ester (Ptox^Dpt) Compound 45 (Figure 10), both of which exhibited inhibitory effects on the proliferation, migration, and aggressiveness of hepatocellular carcinoma cells in vitro and in vivo experiments. Furthermore, in terms of molecular mechanisms, Ptox^Pdp and Ptox^Dpt may inhibit the EMT ability of hepatocellular carcinoma cells through down-regulation of the PI3K/AKT/mTOR pathway, causing a reduction of vimentin expression and an increase of E-cadherin expression.

Glucose metabolism is a key feature of tumor cells, and their proliferation is accompanied by increased demand for energy and metabolic substrates. Many cancers are adapting to these demands through a shift in metabolism termed the Warburg effect, which inhibits oxidative phosphorylation and thus promotes a glucose-to-lactate aerobic fermentation (Vander Heiden et al., 2009). A better insight into cellular metabolism may ultimately lead to superior treatments for human cancers. The effects of PTOX and its derivatives on glucose metabolism have been poorly studied. Tailor et al. (2021) synthesized a new PTOX derivative, SU212 Compound 46 (Figure 10), which exhibited selective anticancer toxicity through direct activation of AMP-activated protein kinase (AMPK). Moreover, this PTOX derivative could regulate the Warburg effect in triple-negative breast cancer cells through the AMPK/hypoxia-inducible factor 1α (HIF-1α) pathway, suggesting the potential research interest of PTOX derivatives in the field of tumor glycolysis.