Abstract

   Colorectal cancer (CRC) is one of the most prevalent cancers due to its
   frequency and high rate of mortality. Polyamine-vectorized anticancer
   drugs possess multiple biological properties. Of these drugs, 9F has
   been shown to inhibit tumor growth and the metastasis of hepatocellular
   carcinoma. This current study aims to investigate the effects of 9F on
   CRC and determine its molecular mechanisms of action. Our findings
   demonstrate that 9F inhibits CRC cell growth by inducing apoptosis and
   cell cycle arrest, and suppresses migration, invasion and angiogenesis
   in vitro, resulting in the inhibition of tumor growth and metastasis in
   vivo. Based on RNA-seq data, further bioinformatic analyses suggest
   that 9F exerts its anticancer activities through p53 signaling, which
   is responsible for the altered expression of key regulators of the cell
   cycle, apoptosis, the epithelial-to-mesenchymal transition (EMT), and
   angiogenesis. In addition, 9F is more effective than amonafide against
   CRC. These results show that 9F can be considered as a potential
   strategy for CRC treatment.

   Keywords: naphthalimide, polyamine, colorectal cancer, p53

1. Introduction

   Colorectal cancer (CRC) is one of the mostmalignant types of cancers
   that have been increasing in prevalence on a global scale. Due to its
   high rate ofmortality, CRC is one of the main causes of cancer deathin
   the world [[42]1]. Patients with CRC are usually diagnosed with distant
   metastasis, and the 5-year survival rate is no more than 10% in
   patients with advanced-stage CRC [[43]2].Over the years,
   variousstrategiesincluding chemotherapy (i.e., cytotoxic and molecular
   targeted agents) have been applied to improve the treatment results of
   patients with CRC [[44]3,[45]4].Despite advances in early detection and
   treatment, the number of patients with CRC will still grow in the
   coming decades [[46]5].Thus, exploringnovel therapies is crucial for
   improving clinical outcomes in patients with CRC.

   As versatile functional compounds, naphthalimides possess many
   therapeutic properties such as anticancer and antiviral activities
   [[47]6,[48]7,[49]8]. Several naphthalimide derivatives have been shown
   to exert many inhibitory effects on cancer cells through various
   mechanisms [[50]9,[51]10,[52]11,[53]12]. Notably, several
   naphthalimides, such as amonafide, mitonafide, and elinafide
   [[54]13,[55]14,[56]15], have been evaluated clinically as promising
   anti-cancer agents. For example, amonafide was the first naphthalimide
   evaluated in clinical trials alone [[57]16,[58]17] and in combination
   with other anti-cancer drugs [[59]18]. In recent years, agents composed
   of a polyamine skeleton have displayed various biological properties
   [[60]19,[61]20,[62]21,[63]22]. Supporting the potential importance of
   the polyamine scaffold, anew spermine-vectorized anticancer drug
   ([64]F14512) has been evaluated as an anticancer candidate in phase I
   clinical trials [[65]23,[66]24,[67]25].

   As a classical tumor suppressor, p53 has been investigated extensively
   and has been known to play a critical role in tumor growth and
   metastasis [[68]26]. In response to stress, p53 acts as a
   transcriptional factor that regulates the expression of downstream
   target genes involved in the cell cycle, apoptosis, DNA damage and
   senescence [[69]27,[70]28]. A correlation has been identified between
   p53 and the death receptor [[71]29,[72]30], which suggests a
   significant role of p53 in death receptor-mediated cell death.
   Metastasis is the main cause of cancer death, and the
   epithelial-to-mesenchymal transition (EMT) is a crucial process for
   metastasis [[73]31,[74]32]. Recently, emerging evidence has
   demonstrated that p53 acts as a negative regulator of EMT via by
   mediating the expression of EMT markers [[75]33,[76]34] such as Snail,
   Twist, Slug and Vimentin [[77]35,[78]36,[79]37,[80]38]. These results
   imply that p53 loss or mutation creates a permissive environment for
   metastasis [[81]39].

   In order to increase the antitumor efficacy and reduce the adverse
   effects of naphthalimide derivates, we developed several
   naphthalimide-polyamine conjugates and found evidence of their
   anticancer activities in experimental models using several cancer types
   [[82]40,[83]41,[84]42]. In the present study, we report that 9F, a
   naphthalene diimide derivative with polyamine moieties, inhibits the
   growth and lung metastasis of CRC in vitro and in vivo. Using RNA-seq,
   9F was identified to induce cell cycle arrest, apoptosis, and oppose
   EMT as well as angiogenesis by activating p53-mediated signaling.

2. Results

2.1. 9F Inhibits Cell Growth and Induces Cell Deathof CRC

   The chemical structure of 9F is shown in [85]Figure 1A. To understand
   the effects of 9F on CRC, we examined the inhibitory effects of 9F on
   several cell lines including the CT-26, HCT116 and HCT8 cells. As
   displayed in [86]Figure 1B, the cell viability of these three cell
   lines was obviously decreased after treatment with 9F for 48 h.
   Meanwhile, 9F inhibited the growth rate of CRC cells to a greater
   extent than amonafide, which was used as a positive control ([87]Figure
   1B). To confirm the anticancer properties of 9F, CRC cell lines (i.e.,
   CT-26 and HCT116) were exposed to 9F at various concentrations for 24
   h, 48 h and 72 h. As shown in [88]Figure 1C, treatment with 9F caused a
   significant inhibition of cell viability in a time- and
   concentration-dependent manner. To investigate whether the inhibitory
   effects of 9F on the growth of CRC cells were sustained beyond three
   days, the anti-proliferative effects of 9F were detected using 2D
   colony formation assays. Treatment of CRC cells with 9Fremarkably
   reduced the number of cell colonies ([89]Figure 1D). Consistently, both
   dual acridine orange/ethidium bromide (AO/EB) staining and dual Annexin
   V/propidium iodide (PI) staining revealed an increased rate of cell
   death in 9F-treated groups, compared to the control groups ([90]Figure
   1E,F). Taken together, these results demonstrated the inhibition of
   cell growth and induction of cell death by 9F in CRC cells.

Figure 1.

   [91]Figure 1
   [92]Open in a new tab

   9F inhibited cell growth and induced cell death of colorectal cancer
   (CRC) cells. (A) The chemical structure of 9F. (B) Cell viability was
   measured in CRC cells treated with various concentrations of 9F and
   amonafide for 48 h. (C) Cell viability was measured in CRC cells
   treated with various concentrations of 9F for 24 h, 48 h and 72 h. (D)
   Representative images (HCT116 cells) and statistics results of colony
   formation assay in CRC cells. CRC cells were treated with 9F at
   different concentrations for 7 days. ** p < 0.01, and *** p < 0.001.
   (E) AO/EB staining was used to detect cell death of CRC cells exposed
   to 9F for 48 h (20×). (F) CT-26 cells and HCT116 cells were treated
   with 9F (1 μM) for 48 h. Cell death was detected using flow cytometry.

2.2. Identification of p53-Mediated Pathway in 9F-Treated CRC Cells

   To decipher the potential mechanisms underlying the inhibitory effects
   of 9F on CRC cells, RNA-seq was carried out to profile changes in the
   transcriptome of HCT116 cells after treatment with 9F. Based on the
   results of MTT assays, cells were treated with 9F (1 μM) for 24 h. To
   analyze the differentially expressed genes (DEGs), the following
   criteria were applied: log2 fold changes (FCs) > 1 and a false
   detection rate (FDR) of less than 0.05. In total, we identified 1217
   genes with significantly altered expression in HCT116 cells treated by
   9F. Among these DEGs, 564 genes were upregulated, and 653 genes were
   downregulated ([93]Figure 2A). Most importantly, the Kyoto Encyclopedia
   of Genes and Genomes (KEGG) pathway enrichment analysis for the DEGs
   displayed thetop 20 pathways that were enriched. Among these pathways,
   signaling cascades affecting the cell cycle, DNA replication, the p53
   signaling pathway, and apoptosis were significantly enriched
   ([94]Figure 2B). Thep53 signaling pathway plays a critical role in the
   regulation of the cell cycle, DNA replication and apoptosis
   [[95]27,[96]28]. Therefore, we speculated that the p53 signaling
   pathway is the main determinant responsible for the inhibitory effects
   of 9F on CRC. We performed gene set enrichment analysis (GSEA), a
   powerful analytical method for interpreting genome-wide expression
   profiles, to test this hypothesis and to obtain an unbiased result. As
   shown in [97]Figure 2C, the GSEA results implied that the significant
   hallmark gene set that was enriched in 9F-treated cells was implicated
   in p53 signaling. In addition, the p53 downstream pathway was also
   enrichedin 9F-treated cells. As a tumor suppressor, p53 exerts its
   functions by mediating the transcription of a series of genes.
   Considering this aspect, DEGs were analyzed using the Reactome
   database. In 9F-treated cells, the genes regulated by p53 are involved
   in cell death, the cell cycle, cellular metabolism and DNA repair
   ([98]Figure 2C). The nuclear accumulation of p53 is required for the
   transcription of genes, and we found that nuclear localization of p53
   increased in 9F-treated CRC cells ([99]Figure 2D). Western blot results
   indicated that 9F increased the protein expression of p53, p21, and
   sestrin 2 (SESN2) in p53-wildtype HCT116 cells ([100]Figure 2E). In
   contrast, 9F failed to activate the expression of p21 and SESN2 in
   p53-null HCT116 cells, thus suggesting that 9F induces the expression
   of p21 and SESN2 by a p53-dependent pathway. These results indicated
   that p53 and its target genes may contribute to the inhibitory effects
   of 9F on CRC cells.

Figure 2.

   [101]Figure 2
   [102]Open in a new tab

   p53 signaling was altered in 9F-treated CRC cells. (A) Cluster heatmap
   of differentially expressed genes in 9F-treated HCT116 cells. Cells
   were treated by 9F (1 μM) for 24 h. (B) KEGG pathway enrichment
   analysis of differentially expressed genes (DEGs) were performed. The
   top20 pathways were shown. (C) Gene set enrichment analysis (GSEA)
   plots correlating with p53 pathway and p53 downstream pathway (left),
   and Reactome analyses of transcriptional regulation by p53 (right). (D)
   Immunofluorescence staining showing localization of p53 in nuclear in
   HCT116 cells treated by 9F for 48 h (63×). (E) Western blot analyses
   showing p53, p21 and Sestrin 2 protein levels in p53-wildtype HCT116
   cells and p53-null HCT116 cells. Cells were treated by 9F for 48 h.
   β-actin was used as loading control.

2.3. 9F Induces Cell Cycle Arrest and Disrupts DNA Replication of CRCCells

   The Reactome database was used to analyze DEGs. As shown in [103]Figure
   3A, the pathways participating in the cell cycle, DNA replication and
   DNA repair were obviously altered in 9F-treated HCT116 cells. Next,
   DEGs were further subjected to GO enrichment analysis, which identified
   the list of genes that were enriched in the cell cycle, the cell cycle
   process, the mitotic cell cycle process, mitotic cell cycle, cell
   division, DNA replication, sister chromatid segregation, the DNA
   metabolic process, nuclear division, and the chromosome segregation
   process ([104]Figure 3B). These biological processes are involved in
   the cell cycle and DNA replication in HCT116 cells following exposure
   to 9F. In line with the above findings, using GSEA, there was a
   significant reduction in expression of genes associated with the cell
   cycle, G2/M checkpoint, DNA replication, and the E2F pathway in HCT 116
   cells after treatment with 9F ([105]Figure 3C). Among these
   dysregulated genes, CDKN1A, MDM2, GADD45A, GADD45B, CDKN2B and TMEM40
   showed increased expression after 9F treatment. In contrast, CDC25A,
   CDC25C, CDC45, CDC6, CCNB1, CCNB2, CCNA2, CCNE2, E2F1, E2F2, WEE1, and
   minichromosome maintenance (MCM) proteins -2 to -7 were rapidly
   downregulated in HCT116 cells after 9F treatment ([106]Figure 3D).
   Having shown that 9F mediates the expression of genes related to the
   cell cycle based on the data from RNA-seq, we questioned whether 9F
   induces cell cycle arrest. As expected, flow cytometry showed that
   treatment with 9F resulted in G2/M arrest among HCT116 cells and CT-26
   cells ([107]Figure 3E). These results were consistent with the GSEA
   results showing that 9F downregulated the expression of genes involved
   in the G2/M checkpoint ([108]Figure 3C). Interestingly, as demonstrated
   by the western blot assay, the protein levels of GADD45A were
   remarkably increased, while MCM4, MCM7, Wee1, Cdc25C, Cyclin A, Cyclin
   B, Cyclin E, and proliferating-cell nuclear antigen (PCNA) protein
   levels were evidently reduced after 9F treatment in HCT116 cells
   ([109]Figure 3F), thus confirming the data of the RNA-seq analysis.
   These results indicated that cell cycle arrest and disruption of DNA
   replication were induced by 9F in CRC cells.

Figure 3.

   [110]Figure 3
   [111]Open in a new tab

   9F induced G2/M cell cycle arrest in CRC cells. (A) Reactome pathway
   database was used to analyse DEGs in 9F-treated HCT116 cells. (B) GO
   enrichment analyses of DEGs in 9F-treated HCT116 cells. (C) GSEA plots
   correlating with cell cycle, DNA replication, G2/M checkpoint and E2F
   targets. (D) The transcript levels of genes related with cell cycle and
   DNA replication from RNA-seq data. (E) CT-26 cells and HCT116 cells
   were exposed to 9F for 48 h. The cells were stained using PI, and the
   cell cycle progression was analyzed by flow cytometry. (F) Western blot
   analyses of cell cycle and DNA replication regulatory proteins
   following 9F treatment for 48 h.

2.4. Both the Mitochondria-Mediated Pathway and the Death Receptor-Induced
Pathway Play an Important Role in 9F-Induced Apoptosis

   Given the induction of cell death by 9F, as detected using flow
   cytometry and high-content screening (HCS), we next sought to explore
   the pathways involved in 9F-induced apoptosis in CRC cells. We observed
   that genes related to apoptosis were roughly activated in 9F-treated
   HCT116 cells ([112]Figure 4A), thus confirming the effect of 9F on
   apoptosis. Similarly, the gene sets regulating cell death induced by
   p53, the TNF-related apoptosis-inducing ligand (TRAIL) pathway and
   death receptors were enriched following 9F treatment ([113]Figure 4A),
   thus providing evidence that the death receptor-mediated pathway
   contributes to 9F-induced apoptosis. As shown in [114]Figure 4B, 9F
   treatment effectively promoted the expression of FAS, TNFRSF10A, and
   TNFRSF10B. Additionally, PIDD1 was increased and BCL2 was reduced in
   HCT116 cells exposed to 9F for 24 h. To characterize the role of p53 in
   the death receptor pathway regulated by 9F in CRC cells, TP53 and other
   genes involved in the death receptor pathway were uploaded into the
   protein-protein interaction (PPI) online tool Search Tool for the
   Retrieval of Interacting Genes (STRING) ([115]Figure 4C). Moreover,
   treatment with 9F increased the expression of Bak, cleaved caspase 3,
   cleaved caspase 9, death receptor 5, and cleaved caspase 8 in a
   dose-dependent manner ([116]Figure 4D). These results indicated that 9F
   may induce apoptosis through intrinsic and extrinsic pathways.

Figure 4.

   [117]Figure 4
   [118]Open in a new tab

   Activation of intrinsic and extrinsic apoptosis pathways accounted for
   9F-induced cell death. (A) GSEA plots correlating with apoptosis, p53
   regulates transcription of cell death genes, TNF-related
   apoptosis-inducing ligand (TRAIL) pathway and extrinsic apoptotic
   signaling pathway via death domain receptors. (B) Heatmap of
   differentially expressed genes involved in apoptosis. (C)
   Protein-protein interaction (PPI) network based on Search Tool for the
   Retrieval of Interacting Genes (STRING) showing the detailed
   interactions of p53 and cell death-related proteins. (D) Western blot
   analyses of Bak, cleaved caspase 8, cleaved caspase 3, cleaved caspase
   9 and death receptor 5 following 9F treatment for 48 h.

2.5. 9F Inhibits Migration and Invasion of CRC Cells

   Metastasis is a poor prognostic sign for patients with CRC, and is a
   positive predictor for CRC-related mortality. Migration and invasion
   are two key steps that occur during metastasis. We thus explored the
   biological efficacy of 9F against the motility of CRC cells using
   modified Boyden chamber assays. As shown in [119]Figure 5A, treatment
   with 9F decreased the migration of CRC cell lines (CT-26 and HCT116) in
   a concentration-dependent manner. The quantification of migrated cells
   confirmed that CRC cells were sensitive to 9F in migration assays.
   During invasion assays, CRC cells had similar responses to 9F
   ([120]Figure 5B). The EMT phenotype facilitates the migration and
   invasion of CRC cells. Thus, we investigated whether 9F might be able
   to regulate the expression of EMT markers. The downregulation of
   β-catenin, Snail 1, Vimentin, and N-cadherin, as well as the
   upregulation of E-cadherin, demonstrated that 9F affected EMT in CRC
   cells ([121]Figure 5C). To identify the function of p53 in EMT in
   9F-treated CRC cells, the STRING database was applied to validate the
   interactions between p53, β-catenin, Snail 1, Vimentin, N-cadherin, and
   E-cadherin ([122]Figure 5D). Taken together, these results demonstrated
   that 9F inhibited EMT, resulting in the suppression of migration and
   invasion in CRC cells.

Figure 5.

   [123]Figure 5
   [124]Open in a new tab

   The abilities of migration and invasion were lost in CRC cells exposed
   to 9F. (A) Cell migration of CT-26 cells and HCT116 cells was analyzed
   by the Boyden chamber assay (20×). ** p < 0.01, and *** p < 0.001. (B)
   Cell invasion of CT-26 cells and HCT116 cells was analyzed by the
   Boyden chamber assay (20×). ** p < 0.01, and *** p < 0.001. (C) Western
   blot analyses of epithelial-to-mesenchymal transition (EMT) markers in
   HCT116 cells following 9F treatment for 48 h. (D) PPI network based on
   STRING showing the detailed interactions of p53 and EMT markers.

2.6. 9F Suppresses Proliferation, Migration and Tube Formation of Endothelial
Cells

   Angiogenesis is essential for tumor growth and metastasis. Therefore,
   angiogenesis is considered as a therapeutic target for the treatment of
   cancer. Based on the RNA-seq data, we observed that genes that suppress
   tumor angiogenesis, including SERPINE1, THBS1, SERPINB5, CD82 and
   ANGPTL4, were upregulated. By contrast, SLIT2, EFNB2, FGF9, and HEY1,
   which promote tumor angiogenesis, were downregulated in 9F-treated
   HCT116 cells ([125]Figure 6A). As shown in [126]Figure 6B, interactions
   between p53 and these genes were observed. Besides, we found that
   treatment with 9F upregulated the expression of p53 and p21 in human
   microvascular endothelial cells (HMEC-1; [127]Figure 6C). These results
   implied that 9F might affect angiogenesis through p53 signaling. To
   validate the sensitivity of endothelial cell lines (HUVEC and HMEC-1)
   to 9F, we first investigated the effects of 9F on the proliferation of
   HUVEC and HMEC-1 cells. As expected, the cell viability of endothelial
   cells decreased when cells were exposed to 9F ([128]Figure 6D), which
   also suggested the toxic effects of 9F on endothelial cells to a
   certain degree. Moreover, we performed a wound-healing migration assay
   to examine the migration properties of endothelial cells. We observed a
   decrease in endothelial migration depending on the concentration of 9F
   ([129]Figure 6E). Finally, in the tube formation assay, 9F caused a
   decrease in the formation of tubular structures ([130]Figure 6F). These
   observations all indicated that 9F inhibited angiogenesis.

Figure 6.

   [131]Figure 6
   [132]Open in a new tab

   9F suppressed proliferation, migration and tube formation of
   endothelial cells. (A) The transcript levels of genes related to
   angiogenesis from RNA-seq data. (B) PPI network based on STRING showing
   the detailed interactions of p53 and the angiogenesis-related genes
   regulated by 9F treatment. (C) Western blot analyses of p53 and p21
   protein in 9F-treated HMEC-1 cells. Cells were treated by 9Ffor 48 h.
   (D) Cell viability was measured in human endothelial cells treated with
   various concentrations of 9F for 48 h. * p < 0.05, ** p < 0.01. (E)
   Cell migration of endothelial cells was analyzed by the wound healing
   assay (10×). *** p < 0.001. (F) Tube formation by endothelial cells was
   assayed after 9F treatment for 8 h (10×). ** p < 0.01, and *** p <
   0.001.

2.7. 9F Suppresses Tumor Growth and Lung Metastasis of CRC In Vivo

   To determine whether 9F suppresses tumor growth in vivo, a xenograft
   model was established. Mice were treated with physiological saline
   (control), 9F (0.2 mg/kg and 0.3 mg/kg) and amonafide (5 mg/kg) by
   intravenous injection. As displayed in [133]Figure 7A, tumor growth in
   treated groups was retarded. Meanwhile, 9F at a high dose (0.3 mg/kg)
   yielded better results than amonafide (5.0 mg/kg). The index of body
   weight of mice demonstrated that there were no dose-associated
   toxicities ([134]Figure 7B), which was further confirmed by
   morphological examination of the livers and lungs of the mice from the
   control and 9F (0.3 mg/kg)-treated group ([135]Figure 7C).
   Subsequently, we assessed the impact of 9F on the lung metastasis of
   CRC. As shown in [136]Figure 7D,E, treatment with 9F and amonafide
   obviously reduced the number of metastasis nodules. Consistent with the
   tumor growth curve, the high dose of 9Fshowed better treatment effects,
   as compared to amonafide, in the lung metastasis model. Taken together,
   these results demonstrated that 9Fwas capable of inhibiting tumor
   growth and metastasis in vivo.

Figure 7.

   [137]Figure 7
   [138]Open in a new tab

   Treatment with 9F inhibited tumor growth and metastasis of CRC in vivo.
   (A) Tumor volumes of mice treated by 9F and amonafide were evaluated on
   the indicated days. Amonafide was used as positive control. * p < 0.05,
   *** p < 0.001. (B) Body weights of mice were measured. (C) H&E staining
   of liver and lung from mice bearing CRC cells after treatment with or
   without 9F (0.3 mg/kg). (D) The representative images of lung
   metastasis nodules from mice bearing CRC cells after treatment with or
   without 9F (0.3 mg/kg). The number of metastasis nodules was recorded.
   Amonafide was used as positive control. ** p < 0.01, and *** p < 0.001.
   (E) The representative images of H&E staining of lung metastasis from
   mice treated with or without 9F (0.3 mg/kg).

3. Discussion

   Previous studies have considered the evaluation of several
   naphthalimides in clinical trials. However, adverse side effects (e.g.,
   dose-limiting myelosuppression) were observed, which limits the
   clinical application of amonafide and its derivatives
   [[139]43,[140]44]. In contrast, 9F has demonstrated lower IC[50]
   profiling against hepatocellular carcinoma, compared to amonafide. Of
   importance, 9F has a lower number of adverse side effects and is safer
   than amonafide [[141]41]. The biological effects and underlying
   mechanisms of 9F against CRC have not been reported. In this study, we
   functionally characterized 9F as more effective than amonafide against
   CRC in vitro and in vivo. Based on RNA-seq, we revealed that the
   activation of the p53 signaling was responsible for the efficacy of 9F
   against CRC.

   We found that 9F increased the expression of p53 and the localization
   of p53 in nuclei ([142]Figure 2). Accumulation of p53, as a nuclear
   transcriptional factor, triggers transcriptional transactivation of its
   downstream genes such as p21 and GADD45, resulting in cell cycle arrest
   [[143]45]. Cell cycle progression is dysregulated in numerous malignant
   diseases. Therefore, modulation of the cell cycle has been considered
   as a potential therapeutic strategy for cancer treatment [[144]46]. We
   have shown that 9F is capable of decreasing the protein expression of
   Wee1, Cdc25C, Cyclin B, and cyclin E, which are related to theG2/M cell
   cycle transition ([145]Figure 3). In response to stress, Gadd45A is a
   key player in G2/M cell cycle arrest. The induction of Gadd45A
   expression confirmed that G2/M cell cycle arrest was induced in
   9F-treated cells. Activation of oncogenes and dysregulation of the cell
   cycle lead to stalled DNA replication forks [[146]47]. Many factors
   participate in the process of DNA replication. Among cell cycle
   regulatory proteins, p21 plays a direct role in inhibiting DNA
   replication [[147]48]. In addition, E2F and PCNA integrate the cell
   cycle with DNA replication [[148]49,[149]50]. In eukaryotic cells, the
   MCM complex (known as DNA replication licensing) controls the
   once-per-cell cycle DNA replication process [[150]51,[151]52].
   Therefore, targeting DNA replication stress is one strategy for cancer
   treatment. Recent advances have indicated that topoisomerase inhibitors
   could induce replicative stress by controlling DNA supercoiling and
   entanglement [[152]53]. Some topoisomerase inhibitors such as
   camptothecin (CPT), which targets Topo I, and doxorubicin and
   mitoxantrone, which target Topo II, have been used for cancer therapy
   in clinical trials [[153]54]. Recently, a series of naphthalimide
   derivatives have been assessed as topoisomerase inhibitors
   [[154]8,[155]55]. In line with these findings, our results implied that
   9F could be considered as a potential candidate for CRC therapy.

   Apoptosis is genetically regulated by the mitochondria-mediated
   intrinsic pathway and the death receptor-induced extrinsic pathway to
   control physiological growth and tissue homeostasis [[156]56]. Current
   cancer therapies primarily trigger apoptosis in cancer cells to exert
   their anticancer effects. Death receptor genes are likely enhanced
   through p53-dependent and -independent mechanisms in response to
   antitumor agents [[157]57,[158]58]. Studies have shown that
   DNA-damaging agents can increase the expression of DR4 [[159]59], which
   is upregulated by p53 through an intronic p53 binding site [[160]60].
   When cells are exposed to genotoxic stress, DR5 was possibly regulated
   in a p53-dependent manner [[161]58]. Naphthalimide–polyamine conjugates
   have been previously shown to induce cell death, partially through the
   death receptor-mediated pathway [[162]61]. In the present study, we
   observed that the expression of PIDD, FAS, DR4, and DR5 were increased
   at mRNA level, while Bcl-2 was reduced in 9F-treated colorectal cells
   ([163]Figure 4). Meanwhile, the treatment of HCT116 cells induced the
   expression of Bak, cleaved caspase 3, cleaved caspase 9, DR 5, and
   cleaved caspase 8. These mechanisms may contribute to 9F-induced
   apoptosis and suggest that 9F-induced cell death occurs through both
   intrinsic and extrinsic pathways. In addition, we will investigate the
   detailed role of death receptors, including DR4 and DR5, in cells
   treated by 9F (and its derivatives) in our future work.

   Cells isolated from p53−/− mice harbor increased the expression of
   Twist, Snai1, Snai2, Vimentin, Zeb1, and Zeb2, and decreased the
   expression of E-cadherin [[164]38]. These results indicated that the
   EMT phenotype was reversed in the absence of p53. Valentini et al.
   found that the accumulation of β-catenin was observed in CRCs with high
   frequency mutations of p53 [[165]62]. Sadot et al. also observed that
   the activation of p53 resulted in the downregulation of β-catenin
   [[166]63], a marker of EMT, which promotes the migration and invasion
   of CRC [[167]64]. Our results indicated that 9F inhibited the migration
   and invasion of CRCs by altering the expression of EMT markers
   ([168]Figure 5). Naphthalimide–polyamine conjugates have been evaluated
   in their ability to prevent the migration and invasion of cancer cells
   and, subsequently, to suppress metastasis in vivo [[169]40,[170]42]. In
   addition, treatment with 9F induced cell cycle arrest and proliferation
   inhibition affects the cell viability, resulting in the partial
   inhibition of EMT. Thus, the effects of topoisomerase inhibitors, such
   as amonafide and its derivatives, on EMT should be investigated under
   lower concentrations (which will not induce cell cycle arrest or DNA
   damage) in future work. Taken together, inhibition of EMT regulated by
   p53 may be responsible for the improved therapeutic response to 9F
   through the suppression of metastasis of CRC in vivo ([171]Figure 7).

   Tumor angiogenesis is a prognostic factor used to characterize CRC
   patient outcome. Emerging studies have revealed that the microvascular
   density of CRC is related to tumor growth and metastasis
   [[172]65,[173]66]. Hence, targeting angiogenesis has been considered as
   an attractive strategy for CRC treatment. To block tumor angiogenesis,
   angiogenesis inhibitors have been developed intensively. These drugs
   range from endogenous angiogenesis inhibitors to monoclonal antibodies
   as well as small molecules. Some of them have been successfully
   integrated into current treatment regimens and have displayed efficacy
   in CRC treatment [[174]67]. Many proteins such as thrombospondins,
   vasohibin, and chondromodulin, have been identified as endogenous
   angiogenesis inhibitors [[175]68,[176]69,[177]70]. Our RNA-seq results
   indicated that 9F treatment induced the transcript levels of
   thrombospondin 1, SERPINB5, CD82, and SERPINE1 ([178]Figure 6), which
   have been reported to display anti-angiogenic activities upon p53
   activation [[179]71]. In this study, we found that endothelial cells
   are sensitive to treatment with 9F, and that expression of p53 and p21
   is upregulated in 9F-treated endothelial cells ([180]Figure 6). The
   inhibitory effects of naphthalimide-polyamine conjugates on
   angiogenesis have rarely been reported. Analyzing the activity of other
   naphthalimide-polyamine conjugates against angiogenesis will be
   necessary to determine whether angiogenesis is involved in the
   suppressive effects of naphthalimide-polyamine conjugates on CRC.

4. Materials and Methods

4.1. Cell Lines and Reagents

   Colorectal cancer cells lines HCT8, CT-26, Human umbilical vein
   endothelia cell (HUVEC) and human microvessel endothelial cell-1
   (HMEC-1) were obtained from the American Type Culture Collection (ATCC,
   Manassas, VA, USA). HCT116 p53−/− and HCT116 p53+/+ were kind gifts
   from Prof. Xiaotao Li, East China Normal University. Colorectal cancer
   cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific,
   Waltham, MA, USA) supplemented with 10% FBS (PAN-Seratech, Aidenbach,
   Germany), streptomycin (100 units/mL), and penicillin (100 units/mL) at
   37 °C in 5% CO[2]. 9F was synthesized in our laboratory.

4.2. MTT Assay

   Cell viability was measured using MTT assay [[181]72]. In brief, 4 ×
   10^3 cells were seeded into 96-well plates and exposed to different
   concentrations of 9F or amonafide for the indicated time. After
   treatment, 50 μL MTT (1 mg/mL) was added followed by incubation at 37
   °C and 100 μL DMSO was added to dissolve the crystal products.
   Absorbance was measured using a microtiter plate reader, and all
   experiments were repeated at least three times. Antibodies were
   purchased from Cell Signaling Technology Inc (Danvers, MA, USA).

4.3. 2D Colony Formation Assay

   The 2D colony formation assay was carried out for measurement of
   clonogenic cell survival. Briefly, 500 cells in 100 μL medium were
   seeded into the wells of 96-well plates. Cells were cultured for 3 days
   and then treated with 9F at various concentrations for another 7 days.
   Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min,
   and stained with crystal violet. Images were taken and colonies were
   counted. All experiments were repeated three times.

4.4. Apoptosis Detection

   AO/EB staining and Annexin V-FITC/PI staining were used to examine the
   apoptosis of colorectal cancer cells as previous description. In brief,
   after cells were treated with 9F for 48 h, cells were stained with
   AO/EB or Annexin V-FITC/PI, and the fluorescence of AO/EB and Annexin
   V-FITC/PI were determined by high content screening (HCS) and flow
   cytometry, respectively (BD Biosciences, San Jose, CA, USA).

4.5. Cell Cycle Assay

   HCT116 cells and CT-26 cells were treated by 9F for 48 h, then
   harvested and washed with PBS. Cells were fixed with 70% cold ethanol
   overnight at 4 °C. Cells were collected and re-suspended with PBS
   containing RNase. After incubation at 37 °C for 30 min, cells were
   stained with PI and analyzed using flow cytometry.

4.6. Immunofluorescence Assay

   HCT116 cells were treated with 9F for 48 h, and fixed by 4%
   formaldehyde. Fixed cells were treated with 0.2% Triton-X 100 and
   blocked using 0.5% FBS. After incubation with the anti-p53 antibody
   overnight at 4 °C, cells were treated with the secondary antibody.
   Then, Hoechst33342 was added to stain the nucleus. Images were taken by
   confocal microscopy (Nikon, Tokyo, Japan).

4.7. Western Bloting Assay

   Cells were treated with 9F at various concentrations for 48 h and
   harvested. Collected cells were washed with cold PBS and lysed using
   RIPA buffer (Beyotime, Shanghai, China). A BCA assay kit was used to
   detect the concentration of proteins [[182]73]. The samples were
   subjected to SDS-loading buffer at 100 °C for 10 min and exposed to
   SDS-PAGE. Proteins were transferred onto PVDF membranes which were
   subsequently blocked by 5% dried skimmed milk. The expression of
   protein was detected using the ECL plus reagents (Beyotime).

4.8. Boyden Chamber Migration and Invasion Assay

   A boyden chamber system was used to assess colorectal cancer cell
   migration and invasion. After starvation for 8 h, cells were seeded to
   the chamber with 8-μm pore. Images were taken after 9F treatment for 24
   h, and migrated cells were recorded. For the invasion assay, the
   chamber was pre-coated with Matrigel.

4.9. Angiogenesis Assay

   A wound healing migration assay and tube formation assay were performed
   to evaluate the effect of 9F on angiogenesis [[183]74]. Endothelial
   cells were cultured in 6-well plates pre-coated with 1% gelatin. Cells
   were scratched with a pipette tip followed by the addition of 9F at
   various concentrations. Images of the wound area were taken after 9F
   treatment for 8 h, and the number of migrated cells was recorded. As
   for the tube formation assay, 96-well plates were pre-coated with
   Matrigel, and 1 × 10^4 endothelial cells per well were seeded. After
   incubation for 8 h, tube structures were photographed and counted.

4.10. Mice, Tumor Model and Treatment

   All animal experiments performed in this article were conducted in
   compliance with the Guide for the Care and Use of Laboratory Animals
   published by Henan University (protocol code: HUSOM-2019-162).
   Six-week-old Balb/c mice were injected subcutaneously with CT-26 cells
   (5 × 10^5 cells per mouse). After 3 days, mice (6 mice/each group) were
   treated with physiologic saline (control), 9F (0.2 mg/kg and 0.3 mg/kg)
   and amonafide (5 mg/kg) via the tail vein for 18 consecutive days. Body
   weight and tumor dimensions were measured performed daily. The tumor
   volume was considered the volume equal to 0.52 × AB^2, where A is the
   long axis and B is the short axis. CT-26 cells (1 × 10^5 cells per
   mouse) were injected via the tail vein to establish lung metastasis
   model. To ensure the establishment of lung metastasis, the tumor was
   incubated for 3 days. Next, mice (5 mice/each group) were treated with
   physiologic saline (control), 9F (0.2 mg/kg and 0.3 mg/kg) and
   amonafide (5 mg/kg) via the tail vein every day. After treatment for 21
   days, mice were anesthetized and euthanized. Lungs were removed, and
   the nodules were counted.

4.11. Hematoxylin and Eosin Staining

   Tissues from the mice were fixed by 4% paraformaldehyde and sectioned
   at 5 μm, haematoxylin and eosin staining was performed according to a
   standard protocol [[184]41].

4.12. RNA-Sequencing and Bioinformatics Analysis

   According to the Wuhan Huada Sequencing Company’s (Wuhan, China)
   instructions, the cDNA library construction, library purification and
   transcriptome sequencing were performed. Gene Ontology (GO) terms,
   Kyoto Encyclopedia of Genes and Genomes (KEGG), REACTOME, and gene set
   enrichment analysis (GSEA) were utilized. Search Tool for the Retrieval
   of Interacting Genes (STRING) was used to predict protein-protein
   interactions (PPI).

4.13. Statistical Analysis

   In this study, data were expressed as mean ± SD from at least three
   separate experiments. Unless otherwise noted, the differences between
   groups were analyzed using Student‘s t-test. p < 0.05 was set to be
   considered statistically significant, * p < 0.05, ** p < 0.01, and ***
   p < 0.001.

5. Conclusions

   Our findings demonstrated that 9F induces apoptosis and cell cycle
   arrestin CRC cells, and suppresses the migration and invasion of CRC
   cells (as well as angiogenesis) in vitro, leading to the inhibition of
   tumor growth and metastasis in vivo. According to bioinformatic
   analyses based on RNA-seq data, we found that 9F regulates the
   expression of key regulators of the cell cycle, apoptosis, the
   epithelial-to-mesenchymal transition (EMT), and angiogenesis through
   p53 signaling. In addition, 9F is more effective than amonafide against
   CRC in vitro and in vivo.

   In summary, we have demonstrated the key role of the p53-mediated
   pathway in examining the therapeutic response of CRC cells to 9F in
   vitro and in vivo. Our findings warrant the further development of 9F
   as a potential therapeutic agent for patients with CRC.

Author Contributions

   Conceptualization, L.G., F.D., S.X. and C.W.; methodology, L.G., F.D.
   and S.W.; software, L.G., C.G., Y.F. and S.W.; validation, S.W., C.G.,
   X.X. and X.L.; formal analysis, S.W., C.G. and F.D.; investigation,
   L.G., F.D., S.W. and Y.W.; resources, Y.W., F.D., S.X. and C.W.; data
   curation, F.D., S.W., C.G. and L.G.; writing—original draft
   preparation, F.D. and L.G.; writing—review and editing, F.D., L.G.,
   C.G., S.X. and C.W.; visualization, L.G. and F.D.; supervision, C.W.
   and S.X.; project administration, F.D., S.X. and C.W.; funding
   acquisition, F.D., S.X. and C.W. All authors have read and agreed to
   the published version of the manuscript.

Funding

   This work was supported by National Science Foundation of China
   (81703004, U1904151, 81772832 and 81573465), China Postdoctoral Science
   Foundation Funded Project (2019M652527), Start-up Fund of Postdoctoral
   Research Program of Henan Province, Postgraduate Education Reform
   Project of Henan Province (2019SJGLX081Y), Young Talents Research and
   Cultivation Program of Henan Medical School (2019011), Innovative
   Research Team (in Science and Technology) in University of Henan
   Province (19IRTSTHN004).

Conflicts of Interest

   The authors declare no competing interest.

References