**1. Introduction**

Cancer is a major health problem worldwide, and the global burden of cancer has caused 10 million deaths in the past year [1]. While recent advances in the development of antitumor agents have contributed to cancer therapy, resistance to chemotherapy has led to recurrence and relapse [2]. In addition, the use of current antitumor agents has been limited due to their toxic and deleterious effects [3]. This calls for the development of novel anticancer agents with high efficacy to combat these issues.

Solid tumors suffer from a harsh microenvironment that has unique features and characteristics such as having areas with compromised endothelium, poor, or avascularized

**Citation:** Abuhijjleh, R.K.; Al Saeedy, D.Y.; Ashmawy, N.S.; Gouda, A.E.; Elhady, S.S.; Al-Abd, A.M. Chemomodulatory Effect of the Marine-Derived Metabolite "Terrein" on the Anticancer Properties of Gemcitabine in Colorectal Cancer Cells. *Mar. Drugs* **2023**, *21*, 271. https://doi.org/10.3390/ md21050271

Academic Editor: Dehai Li

Received: 25 March 2023 Revised: 19 April 2023 Accepted: 22 April 2023 Published: 26 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

areas hindering drugs from reaching their targets, hypoxia in certain areas within the solid tumor, and a high level of acidosis with a noticeable pH gradient along the tumor tissue [4]. All the above features, nonetheless hypoxia, can play a role as drug targets due to the challenges faced with traditional chemotherapies. Oxygen levels in addition to the availability of nutrients differ drastically during the development of tumor cells through angiogenesis, and the recruitment of leukocytes and fibroblasts. Therefore, hypoxia can affect gene expressions, signaling pathways, many metabolic reactions, and the response to stress as well as the response to cytotoxic drugs [5]. Anticancer agents target various survival/death mechanisms or pathways in cancer cells, including angiogenesis, cell cycle regulation, apoptosis, and autophagy [6–9].

The secondary marine metabolite (+)-terrein was first isolated from *Aspergillus terreus* in 1935 and subsequently drew significant attention due to its various bioactivities, including anticancer properties [10]. Promising studies showed that terrein inhibits angiogenin production and secretion [11,12], induces cell cycle arrest [13,14] and apoptosis [15], and inhibits cell proliferation [16]. There is also evidence that terrein has anti-inflammatory activity that is mediated via inactivating the nuclear factor kappa B (NF-kB) signaling pathway. This occurs because of various mechanisms, most notably the inhibition of p60/p50 heterodimer translocation into the nucleus and the DNA-binding activity of the p65 subunit [12,17]. The NF-kB pathway has been implicated in various types of cancer due to its role in regulating apoptosis [18], and its abnormal activation can lead to malignant tumors and oncogenesis [19]. Yet, one of the major obstacles to using terrein on a large scale is the very low yield of this compound from different marine sources [20]. In addition, terrein and other marine-derived secondary metabolites are known for their abundance in diverse geographical distribution and their unique chemical structure [21].

Gemcitabine is the standard drug of choice for locally advanced and metastatic pancreatic cancer [22]. However, it is frequently associated with treatment failure due to intrinsic or acquired resistance. The failure of achieving good clinical outcomes in terms of survival could partially be associated with the hypo-vascularized and dense tumor stroma, and therefore, poor drug penetration and hypoxia [23]. Most patients acquire resistance after weeks of treatment, resulting in poor survival. Gemcitabine resistance can be either intrinsic or acquired and can result from molecular and cellular changes, such as nucleotide metabolism, apoptosis pathway suppression [24], ABC transporter protein overactivation/over-expression [25], activation of the cancer stem cells CSCs [26], activation of the epithelial-to-mesenchymal transition pathway (EMT) [27], and extracellular signal-regulated protein kinase (ERK) 1/2 overactivity [28]. Gemcitabine resistance is also associated with multiple genetic and epigenetic abnormalities. Changes in one or a few genes remain crucial for maintaining drug resistance, cell survival, and malignant phenotype [22]. There is evidence to indicate that NF-κB [29–32], AKT [29,33,34], MAPK [34,35], and HIF-1α pathways [36] are directly related to the resistance of gemcitabine in vitro and in vivo models.

Metabolomics is considered one of the best approaches to studying the effectiveness of drugs towards cancer [37] as well as assessing the reasons behind developing resistance toward drugs [38]. In this study, a metabolomic analysis has been conducted to compare the effect of terrein, gemcitabine, and a combination of both drugs on colorectal cells under normoxic as well as hypoxic conditions. 1HNMR spectroscopy was utilized in combination with chemometric statistical methods to compare the extracellular metabolites after each treatment.

Herein, we evaluated the interaction between terrein and gemcitabine in colorectal cancer cells under normal and hypoxic conditions in terms of a potential chemomodulatory effect and mutual intra-tumoral metabolic influence.

#### **2. Results**

#### *2.1. Cytotoxicity Assessment*

To study the effect of Terr on the cytotoxic profile of GCB in colorectal cancer cell lines (HCT-116, HT-29, and SW620) under normoxic and hypoxic conditions, the viability dose–response curves of both agents, alone and in combination, were assessed using the Emax model as described in the Materials and Methods section. The IC50s of either agent, alone or in combination with their CI indices, are summarized in Table 1.

**Table 1.** Combination analysis for GCB and Terr against HCT-116, HT-29, and SW620 colorectal cancer cell lines. (N) indicates normoxia, and (H) indicates hypoxia.


In the HCT-116 cells, GCB exerted potent cytotoxic activity despite a resistant fraction of 38.14 ± 1.4% in the normoxia condition; viability started to drop significantly (*p* < 0.05) from the control value at 0.03 μM and 0.01 μM in normoxia and hypoxia, respectively. The cellular log kill was gradual in profile with IC50 of 0.19 ± 0.028 μM and 0.01 ± 0.002 μM in normoxia and hypoxia, respectively. Terr exerted abrupt cytotoxic activity with increasing concentration; viability started to drop significantly at 100 μM in both conditions. The cellular log kill showed IC50 of (75.22 ± 0.97 μM and 20.26 ± 2.89 μM) in normoxia and hypoxia, respectively. An equitoxic combination of Terr with GCB improved the cytotoxic profile of GCB in the HCT-116 cell line in normoxia, decreasing the resistant fraction to 24.40 ± 1.22%; however, it did not improve the cytotoxic profile of GCB in hypoxia with an increased resistant fraction to 41.02 ± 2.33%. More so, the IC50 of GCB after combination with Terr decreased significantly compared to the single GCB treatment in normoxia and increased in hypoxia (Supplementary Figure S1A,B). The calculated CI values for GCB with Terr were 0.129 and 1.779 in normoxia and hypoxia, respectively. These CI values are indicative of synergistic interaction characteristics in the HCT-116 cell line under normoxic conditions and an antagonistic interaction under hypoxic conditions (Table 1).

For the HT-29 cells, GCB had a resistant fraction of 19.87 ± 12.0% and 42.64 ± 2.10% in normoxia and hypoxia, respectively; viability dropped significantly (*p* < 0.05) compared to the control cells at 0.01 μM and 0.03μM in normoxia and hypoxia, respectively (Supplementary Figure S2A,B). The IC50s of GCB were 0.01 ± 0.006 μM and 0.04 ± 0.004 μM in normoxia and hypoxia, respectively. The viability of the cells treated with Terr started to drop significantly (*p* < 0.05) from the concentration of 30 μM in normoxia and 100 μM in hypoxia. The IC50s of Terr were 56.24 ± 11.39 μM and 83.30 ± 4.41 μM in normoxia and hypoxia, respectively. An equitoxic combination of Terr with GCB did not improve the cytotoxic profile of GCB; however, it increased the resistant fractions to 46.45 ± 2.76% and 66.74 ± 6.84% in normoxia and hypoxia, respectively. The IC50 of GCB after the combination with Terr significantly increased compared to the single GCB treatment in normoxia and hypoxia. Yet, the calculated CI values for GCB with Terr were 2.318 and 7.277 in normoxia and hypoxia, respectively. These CI values are indicative of antagonistic interaction characteristics in the HT-29 cell line under both oxygen conditions.

With respect to SW620 cells, the resistant fraction after treatment with GCB was 32.62 ± 1.78% and 32.59 ± 0.28% in normoxia and hypoxia, respectively; viability dropped significantly (*p* < 0.05) at 0.3 μM in both normoxia and hypoxia. The IC50 of GCB was 0.21 ± 0.0003 μM, and 0.20 ± 0.008 μM in normoxia and hypoxia, respectively. The viability of the cells treated with Terr dropped significantly (*p* < 0.05) from the control value at 100 μM in both normoxia and hypoxia. The IC50 of Terr was 72.28 ± 1.35 μM, and 59.82 ± 8.50 μM in normoxia and hypoxia, respectively. An equitoxic combination of Terr with GCB improved the cytotoxic profile of GCB, keeping the resistant fraction at 32.30 ± 3.56% and 48.51 ± 2.97% in normoxia and hypoxia, respectively. IC50 of GCB after combination with Terr decreased compared to single GCB treatment in normoxia and hypoxia (Supplementary Figure S3A,B). The calculated CI values for GCB with Terr were 0.092 and 0.142 in normoxia and hypoxia, respectively. These CI values are an indicator of synergistic interaction characteristics in the SW620 cell line under both oxygen conditions.

### *2.2. The Influence of Terr on GCB-Induced Apoptotic Cell Death in Colorectal Cell Lines (HCT-116, HT-29, and SW620)*

The tested colorectal cancer cells were exposed to the predetermined IC50 for 24 h and 48 h and stained with annexin V-FITC/PI. In HCT-116, only the combination treatment (GCB + Terr) significantly induced apoptosis after 24 h and 48 h of exposure (0.51 ± 0.22% and 9.59 ± 0.85%, respectively) compared to the control untreated cells (0.133 ± 0.05% and 3.99 ± 0.37%, respectively) (Figure 1A,D). Single treatments showed no significant difference in terms of apoptosis compared to the control untreated cells. Alternatively, GCB alone induced significant necrosis after 24 h of exposure (14.77 ± 1.06%) compared to the control untreated cells.

In the HT-29 cells, the GCB treatment significantly induced apoptosis after 24 h and 48 h of exposure (8.29 ± 0.92% and 6.06 ± 0.069%, respectively) compared to the control untreated cells (1.24 ± 0.21% and 1.26 ± 0.11%, respectively). The combination treatment (GCB + Terr) was found to significantly decrease apoptosis after 24 h and 48 h of exposure when compared to GCB alone (10.5 ± 0.45% and 10.2 ± 0.09%, respectively). On the other hand, there was no significant difference between GCB and Terr in terms of apoptosis at both time points (Figure 1B,E,H).

In the SW620 cells, treatment with GCB significantly induced apoptosis after 24 h and 48 h of exposure (6.84 ± 0.47% and 2.1 ± 0.13%, respectively) compared to the control untreated cells (1.61 ± 0.28% and 2.24 ± 0.01%, respectively) (Figure 1C,F,I). The combination treatment (GCB + Terr) also increased apoptosis significantly after 24 h and 48 h of exposure (2.97 ± 0.91% and 2.84 ± 0.16%, respectively) when compared to the control untreated cells or GCB treatment alone. The combination treatment (GCB + Terr) induced significant necrosis after 24 h of exposure compared to the control untreated cells (5.17 ± 0.53% and 1.77 ± 0.17%, respectively). This effect was carried forward and influenced the total cell death, where the combination treatment (GCB + Terr) showed a significant increase in total cell death (14.14 ± 1.4%) compared to the control untreated cells (5.38 ± 0.19%) as well as single treatments (GCB or Terr).

To further confirm the flowcytometric apoptosis-driven results, we examined apoptotic regulator genes using the RT-qPCR technique and calculated their fold changes after treatment under normoxic and hypoxic conditions. In HCT-116, the antiapoptotic gene, BCL2, was over-expressed by 25-fold when the cells were treated with terrein alone under normoxic conditions. The same gene showed no significant change in expression after the combination treatment (GCB + Terr). The rest of the treatments in both normoxic and hypoxic conditions resulted in the under-expression of BCL2. Similarly, the apoptosis inhibitor gene, BIRC5, was under-expressed in all treatments in both oxygen conditions. On the other hand, the expression of the tumor suppressor gene, TP53, was not affected after treatment with terrein in normoxia but was under-expressed in all other treatments in both conditions. Similarly, the apoptotic FOXO3 gene showed significant over-expression by 11-fold in normoxia when the cells were treated with terrein. FOXO3 showed no change in

expression when the cells were treated with the combination treatment (GCB + Terr) under hypoxic conditions. The rest of the treatments in both conditions resulted in the underexpression of FOXO3 (Figure 2A,B). In HT-29, all genes tested for apoptosis were underexpressed for all treatments under both normoxia and hypoxia (Figure 2C,D). Similarly, in the SW620 cells, all genes that were tested for apoptosis were under-expressed with all treatments under both normoxia and hypoxia (Figure 2E,F).

**Figure 1.** Programmed cell death (apoptosis) after treatment with Terr, GCB, and their combination for 24 h and 48 h. The cells were stained with annexin V-FITC/PI and different cell populations were plotted as a percentage of total events. HCT-116 under normoxia at 24 h and 48 h (**A**,**D**,**G**), HT-29 under normoxia at 24 h and 48 h (**B**,**E**,**H**), and SW620 under normoxia at 24 h and 48 h (**C**,**F**,**I**). Data are presented as mean ± SD; *n* = 3. \* Significantly different from control group.

**Figure 2.** Fold change of apoptosis regulator genes after treatment with Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates underexpression, above 2.0 indicates over-expression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 at 24 h under normoxia and hypoxia (**A**,**B**), HT-29 at 24 h under normoxia and hypoxia (**C**,**D**), and SW620 at 24 h under normoxia and hypoxia (**E**,**F**). The data are presented as mean fold change ± SD.

Caspase-3 is crucial in the apoptosis process and is considered the executioner active caspase family member; its concentration indicates the actual progression of apoptosis. Herein, active caspase-3 was increased in HCT-116 in response to all single and combined treatments after 24 h and 48 h as well (Figure 3A). However, it was significantly increased in HT-29 when treated with the terrein and GCB combination for 24 and 48 h (Figure 3B). Similarly, the combination of terrein and GCB activated caspase-3 after 48 h only. Surprisingly, GCB induced the activation of caspase-3 after 24 h and 48 h as well (Figure 3C).

**Figure 3.** Caspase-3 level. The active caspase-3 concentration after treatment with Terr, GCB, and their combination in HCT-116 cells (**A**), HT-29 cells (**B**), and SW-620 cells (**C**) for 24 h. The data are presented as mean fold change ± SD. \* Significantly different from control untreated cells.

### *2.3. The Effect of Terr on the Autophagic Cell Death of Colorectal Cell Lines Treated with GCB*

In the HCT-116 cells, GCB induced a significant increase in autophagic cell death by 54.63% compared to the control untreated cells after 24 h under normoxic conditions. Surprisingly, not only Terr but also the combination of GCB + Terr had no significant autophagic effect (Figure 4A,D,G).

In the HT-29 cells, only a combination of GCB + Terr for 48 h induced significant autophagic cell death by 38.18% increase in acridine orange-fluorescent signal compared to the control untreated cells (Figure 4B,D,H).

In the SW620 cells, after 24 h of exposure to the treatment, GCB induced a significant increase in autophagic cell death by 87.62% when compared to the control untreated cells. There was no significant difference between GCB and Terr as single treatments. However, combination treatment (GCB + Terr) induced a significant decrease in autophagic cell death when compared to GCB alone by 35.58%. After 48 h of treatment, the combination treatment induced a significant increase in autophagic cell death by 36.79% when compared to the control untreated cells. However, the combination treatment induced no significant difference when compared with GCB alone (Figure 4C,F,I).

To further confirm flowcytometric-driven results, we examined autophagy regulator genes (ATG5 and Beclin-1) using the RT-qPCR technique and calculated their fold changes after treatment under normoxic and hypoxic conditions. The autophagic-forming vesicle regulator gene, ATG5, was over-expressed when HCT-116 cells were treated with terrein under normoxic conditions by 12-fold. However, it was significantly under-expressed with the other treatments in both oxygen and hypoxia conditions. The golden autophagy standard gene, Beclin-1, was under-expressed in response to all treatments in both oxygen and hypoxia conditions (Figure 5A,B). In HT-29 and SW620, both genes tested for autophagy were under-expressed for all treatments under both normoxia and hypoxia (Figure 5C–F).

**Figure 4.** Induction of programmed cell death (autophagy) by Terr, GCB, and their combination for 24 h and 48 h. The cells were stained with acridine orange. The Average Net Fluorescent Intensity (NFI) values were plotted and compared to control cells. HCT-116 after 24 h and 48 h (**A**,**D**,**G**), HT-29 after 24 h and 48 h (**B**,**E**,**H**), and SW620 after 24 h and 48 h (**C**,**F**,**I**). Data are presented as mean ± SD; *n* = 3. \* Significantly different from control untreated cells.

**Figure 5.** Fold change of autophagy regulator genes by Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates over-expression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 cells under normoxia and hypoxia (**A**,**B**), HT-29 cells under normoxia and hypoxia (**C**,**D**), and SW620 cells under normoxia and hypoxia (**E**,**F**). The data are presented as mean ± SD.
