**6. Salograviolide A and inflammation: Mechanisms and targets**

Natural products derivatives have contributed over the last 25 years to approximately one fourth of the anti-inflammatory drugs used in the clinic (Newman & Cragg, 2007). In 2008, 18 additional natural product derived drugs were being tested at different clinical stages (Harvey, 2008). Terpenoids including sesquiterpene lactones have also reached clinical trials as potential anti-inflammatory agents. For instance, andrographolide; a labdane diterpenoid derived from the plant *Andrographis paniculata* of the Acanthacaea family, reached phase II clinical trials for rheumatoid arthritis. The sesquiterpene lactone parthenolide that made it to phase I cancer clinical trials, has also reached phase II and III clinical trials for the treatment of allergic contact dermatitis (refer to http://www.clinicaltrials.gov/).

Drugs with anti-inflammatory activities have common targets and modes of action that lead to the downregulation of the signs of inflammation as well as the chemical mediators controlling the mechanisms of inflammation. Elements of the complement system, angiogenic factors, prostaglandins, cytokines such as interleukins and matrix metalloproteinases (MMPs) are some of the most common inflammatory mediators.

Prostaglandins play a role in the modulation of blood flow. They are derived from the arachidonic acid due to the action of two prostaglandins H synthases isoforms known as the cyclooxygenases 1 and 2 (COX-1 and COX-2). COX-1 is constitutively expressed in different tissues while COX-2 is induced by inflammatory stimuli and is therefore thought to be the only isoform involved in propagating the inflammatory response (Larsen & Henson, 1983).

IL-6 secreted by the macrophages releases proteinases and elastases which bind to IL-6 receptor and generate signals implicated in humoral inflammation (Heinrich et al., 2003). IL-1, on the other hand, induces the mobilization of the arachidonic acid and its metabolism into prostaglandins thus contributing to cellular inflammation. It was also shown that IL-1 induces the synthesis of COX-2 through the activation of the nuclear factor Kappa B (NF-κB) transcription factor (Larsen & Henson, 1983). NF-κB is a key modulator of inflammation and is implicated in inflammation-induced tumor formation as well (Karin & Greten, 2005). It is

Salograviolide A: A Plant-Derived Sesquiterpene

Lactone with Promising Anti-Inflammatory and Anticancer Effects 381

and with calcium induced differentiation in keratinocytes, respectively were increased (Hochhauser, 1997; Di Cunto et al., 1998, as cited in Ghantous et al., 2007). Moreover, p21 proteins were differentially regulated: they were upregulated in the presence of the crude extract which was consistent with the observed G0/G1 cell cycle arrest, whereas their upregulation in the presence of Sal A was found to be transient. This transient upregulation of p21 has been reported as critical for its role in differentiation (Di Cunto et al., 1998).

Fig. 4. Anti-inflammatory cascade triggered by Sal A. Details of the mechanism are

In addition to cell cycle modulation, characteristic signs of apoptosis such as the partial and complete condensation of the chromatin were noted in the presence of Sal A. Furthermore, the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein Bcl-2 was found to be elevated. Bax and Bcl-2 have counteracting roles regarding the mitochondrial membrane permeabilization and thus the high ratio of Bax to Bcl-2 reflects the permeabilization of the mitochondrial membrane to release factors such as cytochrome c and the apoptosis inducing factor that will induce cell death. In conjunction, a considerable amount of ROS accumulated in the cells in the presence of Sal A. In fact, the accumulation of ROS was even shown to precede the growth inhibition and indicated an oxidant role of Sal A in these cells. Finally, the crude extract and Sal A had a contradictory regulatory effect on NF-κB. The crude plant extract decreased in a dose-dependent manner the binding of NF-κB to the DNA without

explained in section 6 above.

known to promote the expression of target genes of the inflammation response such as interleukins, COX-2, and inducible nitric-oxide synthase (iNOS) (Mazor et al., 2000).

Finally, components of the MMP family have been reported to act in wound healing and embryogenesis (Mainardi et al., 1991). Gelatinase A or MMP-2 (72 KDa) and gelatinase B or MMP-9 (92 KDa) have been identified as pro-inflammatory agents. They enable the digestion of components of the basement membrane; a function that is referred to as gelatinolysis (Birkedal-Hansen et al., 1993).

Sal A and *C. ainetensis* water extract were shown to modulate some of these major players of the inflammatory response. They both inhibit IL-6 expression (Talhouk et al., 2008) and IL-1 induced COX-2 expression by interfering with their synthesis (Al-Saghir et al., 2009). Only the effect of the water extract on the expression levels of the gelatinases A and B was assessed. The results indicate that the water extract decreased the expression of both proteins with preferential inhibition of gelatinase B 9 h post treatment with endotoxin (Talhouk et al., 2008). Similarly, only the effect of Sal A on the NF-κB signaling was investigated. NF-κB is composed of the two subunits p50 and p65 and is only active after translocation of the subunits into the nucleus and their dimerization. In normal conditions, NF-κB is inactive due to its retention in the cytoplasm by the inhibitor of NF-κB (IκB). Proinflammatory stimuli such as IL-1 cause the phosphorylation of the IκB by the inhibitor of NFκB- β (IKK). The phosphorylation in return leads to IκB degradation enabling thus the translocation to the cytoplasm and the dimerization of the NF-κB subunits, their binding to target DNA sequences, initiation of transcription and activation of the NF-κB transduction pathway (Baeuerele & Baltimore, 1996).

It was demonstrated that Sal A inhibits the NF-κB activation by two mechanisms. The first involved the stabilization of IκB, since combination treatment of Sal A with IL-1 decreased the degradation of IκB observed in response to treatment with IL-1 alone. The second involved inhibiting NF-κB translocation and binding to DNA, since the incubation of cells with Sal A after IL-1 addition (hence after IκB degradation) still caused a reduction in the activity of NF-κB (Fig. 4) (Al-Saghir et al., 2009).

#### **7. Salograviolide A and cancer: Mechanisms and targets**

The mechanism behind the anticancer activity of sesquiterpene lactones has been extensively investigated. For example, parthenolide was shown to induce cell cycle arrest, promote cell differentiation and trigger apoptosis (Pajak et al., 2008). The molecular basis for parthenolide activity in cancer cells include among others, inhibiting NF-κB and stimulating apoptosis by accumulation of reactive oxygen species (ROS) as well as by regulating the levels of anti-apoptotic and pro-apoptotic proteins (Pajak et al., 2008). Interestingly, parthenolide showed a synergistic effect when used in combination with paclitaxel and increased apoptosis in breast cancer cells (Patel et al., 2000).

As mentioned earlier, *C. ainetensis* crude extract and Sal A were tested against skin, colon and blood cancer cell lines and showed selective antineoplastic effects with no cytotoxicity to normal cells. At the cellular level, *C. ainetensis* crude extract induced G0/G1 cell cycle arrest in neoplastic epidermal cells while Sal A increased the population in Pre-G1. In accordance with this finding, the levels of cyclin D1 whose activity is required for the G1/S transition were reduced (Li et al., 2011). On the other hand, the levels of the tumor suppressors p16 and p21 which are associated with greater susceptibility to chemotherapy

known to promote the expression of target genes of the inflammation response such as

Finally, components of the MMP family have been reported to act in wound healing and embryogenesis (Mainardi et al., 1991). Gelatinase A or MMP-2 (72 KDa) and gelatinase B or MMP-9 (92 KDa) have been identified as pro-inflammatory agents. They enable the digestion of components of the basement membrane; a function that is referred to as

Sal A and *C. ainetensis* water extract were shown to modulate some of these major players of the inflammatory response. They both inhibit IL-6 expression (Talhouk et al., 2008) and IL-1 induced COX-2 expression by interfering with their synthesis (Al-Saghir et al., 2009). Only the effect of the water extract on the expression levels of the gelatinases A and B was assessed. The results indicate that the water extract decreased the expression of both proteins with preferential inhibition of gelatinase B 9 h post treatment with endotoxin (Talhouk et al., 2008). Similarly, only the effect of Sal A on the NF-κB signaling was investigated. NF-κB is composed of the two subunits p50 and p65 and is only active after translocation of the subunits into the nucleus and their dimerization. In normal conditions, NF-κB is inactive due to its retention in the cytoplasm by the inhibitor of NF-κB (IκB). Proinflammatory stimuli such as IL-1 cause the phosphorylation of the IκB by the inhibitor of NFκB- β (IKK). The phosphorylation in return leads to IκB degradation enabling thus the translocation to the cytoplasm and the dimerization of the NF-κB subunits, their binding to target DNA sequences, initiation of transcription and activation of the NF-κB transduction

It was demonstrated that Sal A inhibits the NF-κB activation by two mechanisms. The first involved the stabilization of IκB, since combination treatment of Sal A with IL-1 decreased the degradation of IκB observed in response to treatment with IL-1 alone. The second involved inhibiting NF-κB translocation and binding to DNA, since the incubation of cells with Sal A after IL-1 addition (hence after IκB degradation) still caused a reduction in the

The mechanism behind the anticancer activity of sesquiterpene lactones has been extensively investigated. For example, parthenolide was shown to induce cell cycle arrest, promote cell differentiation and trigger apoptosis (Pajak et al., 2008). The molecular basis for parthenolide activity in cancer cells include among others, inhibiting NF-κB and stimulating apoptosis by accumulation of reactive oxygen species (ROS) as well as by regulating the levels of anti-apoptotic and pro-apoptotic proteins (Pajak et al., 2008). Interestingly, parthenolide showed a synergistic effect when used in combination with paclitaxel and

As mentioned earlier, *C. ainetensis* crude extract and Sal A were tested against skin, colon and blood cancer cell lines and showed selective antineoplastic effects with no cytotoxicity to normal cells. At the cellular level, *C. ainetensis* crude extract induced G0/G1 cell cycle arrest in neoplastic epidermal cells while Sal A increased the population in Pre-G1. In accordance with this finding, the levels of cyclin D1 whose activity is required for the G1/S transition were reduced (Li et al., 2011). On the other hand, the levels of the tumor suppressors p16 and p21 which are associated with greater susceptibility to chemotherapy

interleukins, COX-2, and inducible nitric-oxide synthase (iNOS) (Mazor et al., 2000).

gelatinolysis (Birkedal-Hansen et al., 1993).

pathway (Baeuerele & Baltimore, 1996).

activity of NF-κB (Fig. 4) (Al-Saghir et al., 2009).

**7. Salograviolide A and cancer: Mechanisms and targets** 

increased apoptosis in breast cancer cells (Patel et al., 2000).

and with calcium induced differentiation in keratinocytes, respectively were increased (Hochhauser, 1997; Di Cunto et al., 1998, as cited in Ghantous et al., 2007). Moreover, p21 proteins were differentially regulated: they were upregulated in the presence of the crude extract which was consistent with the observed G0/G1 cell cycle arrest, whereas their upregulation in the presence of Sal A was found to be transient. This transient upregulation of p21 has been reported as critical for its role in differentiation (Di Cunto et al., 1998).

Fig. 4. Anti-inflammatory cascade triggered by Sal A. Details of the mechanism are explained in section 6 above.

In addition to cell cycle modulation, characteristic signs of apoptosis such as the partial and complete condensation of the chromatin were noted in the presence of Sal A. Furthermore, the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein Bcl-2 was found to be elevated. Bax and Bcl-2 have counteracting roles regarding the mitochondrial membrane permeabilization and thus the high ratio of Bax to Bcl-2 reflects the permeabilization of the mitochondrial membrane to release factors such as cytochrome c and the apoptosis inducing factor that will induce cell death. In conjunction, a considerable amount of ROS accumulated in the cells in the presence of Sal A. In fact, the accumulation of ROS was even shown to precede the growth inhibition and indicated an oxidant role of Sal A in these cells. Finally, the crude extract and Sal A had a contradictory regulatory effect on NF-κB. The crude plant extract decreased in a dose-dependent manner the binding of NF-κB to the DNA without

Salograviolide A: A Plant-Derived Sesquiterpene

mitochondria.

standard clinical drugs.

**8. Conclusion and future direction** 

chronic inflammation (Colotta F. Et al., 2009).

Lactone with Promising Anti-Inflammatory and Anticancer Effects 383

Fig. 5. Anticancer cascade triggered by the combination of Sal A and TNP in colon cancer cells. The combination caused increase in ROS and activation of MAPK molecules, ERK, JNK and p38 leading to apoptosis. It is still unclear whether apoptosis is associated with the

Cancer progression is characterized by seven hallmarks: sustaining proliferative signaling, insensitivity to growth suppressors, evading apoptosis, acquisition of replicative immortality, induction of angiogenesis, activation of invasion and metastasis and finally

In a nutshell, the evidence that has been collected so far by multiple investigators suggests that Sal A is a promising compound for cancer drug discovery for it acts at least on three cancer hallmarks: it upregulates tumor suppressors, triggers apoptosis and downregulates the mediators of inflammation. Its selectivity toward tumor cells and ability to target multiple pathways involved in inflammation and cancer imply that this compound is unlikely to have a single target that is responsible for its biological activities. Although a large body of information supports the role of Sal A against cancer and inflammation, there are yet no studies assessing its toxicology profiles or its absorption, distribution and metabolism in animals and humans. Such studies are warranted to better determine its potential for future applications in the clinical setting whether alone or in combination with

modulation of p21, p53, and cyclin B1 proteins or the release of cytochrome c from

affecting the expression level of IκB, whereas Sal A increased it. It is worth noting that in cancer, the role of NF-κB activation or inhibition is in itself conflicting. NF-κB can be best described as a double edged sword for its activation can promote tumorigenesis (by inducing inflammatory mediators) or promote differentiation and thus inhibit tumorigenesis (Seitz et al., 1998, as cited in Ghantous et al., 2007).

In addition, the crude extract was assessed in human colon cancers and showed both an increase in p21 protein level of expression and in the ratio of Bax to Bcl-2 proteins. In colon cancer, cyclin B1 levels were decreased by the extract, while they were found to be unaffected by neither Sal A nor the extract in skin cancer. Cyclin B1 decrease is important for the exit from mitosis and for the cytokinesis (Takizawa & Morgan, 2000, as cited in El-Najjar et al., 2007). Another protein which was differentially modulated in skin *vs* colon cancer is p53. In colon cancer, the crude extract increased the expression levels of the p53 protein, while the levels were unaffected in skin cancer.

In leukemic cells, similarly to what was shown with the other two types of cancers, the extract induced pre-G1 cell cycle arrest, increased the levels of p53 and p21 as well as the ratio of Bax to Bcl-2, and decreased cyclin D1 levels. In addition, the secretion of the vascular endothelial growth factor (VEGF) was significantly decreased by Sal A and the crude extract, adding VEGF to the list of targets of Sal A (El-Sabban, unpublished findings). Finally, Sal A was tested in combination with TNP against human colon cancer cell lines (Gali-Muhtasib, unpublished findings). At low concentrations, Sal A and TNP induced G2/M cell cycle arrest when tested separately. At the same low concentrations, the combination of Sal A and TNP synergistically inhibited tumor growth and triggered apoptosis. ROS, which increased by a factor of 25 upon combination treatment, were found to be responsible for the synergistic-induced cell death. The pretreatment of the colorectal cancer cells with the antioxidant N-acetyl-L-cysteine (NAC), which diminishes the intracellular ROS, reversed the synergistic anti-proliferative effect and protected the cells from apoptosis and therefore confirmed the implication of ROS in the induction of apoptosis. Moreover, p38 kinase, the extracellular signal regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) of the mitogen-activated protein kinases (MAPK) pathway were found to be phosphorylated and thus induced after the combination treatment. Literature strongly advocates for the MAPK pathway implication in ROS induced cell death (Zhang et al., 2003). Using specific inhibitors of both ERK (usually involved in mitogenic signals and cellular proliferation), and of p38 (associated with stress along with JNK and commonly known as the stress activated protein kinases (Lewis et al., 1998)) abolished the apoptotic synergistic effect of the combination treatment and emphasized the pathway's association to cell death. Further studies with Sal A and TNP suggested a cross-talk between ROS, JNK and Bcl-2 family in the induction of apoptosis. ROS accumulation was found to induce Bax relocalization to the mitochondrial membrane on the one hand and to decrease the ani-apoptotic Bcl-2 expression levels on the other (Gali-Muhtasib, unpublished findings). Bcl-2 decrease was also associated with a maximal JNK activation which suggested that JNK might play a role in further inactivation of Bcl-2. In accordance with our findings several studies have reported the involvement of JNK but not ERK nor p38 in the phosphorylation and inactivation of Bcl-2 (Srivasta et al., 1999).

Taken together these results indicate a promising chemotherapeutic effect for using Sal A alone or in combination against various cancer types.

affecting the expression level of IκB, whereas Sal A increased it. It is worth noting that in cancer, the role of NF-κB activation or inhibition is in itself conflicting. NF-κB can be best described as a double edged sword for its activation can promote tumorigenesis (by inducing inflammatory mediators) or promote differentiation and thus inhibit

In addition, the crude extract was assessed in human colon cancers and showed both an increase in p21 protein level of expression and in the ratio of Bax to Bcl-2 proteins. In colon cancer, cyclin B1 levels were decreased by the extract, while they were found to be unaffected by neither Sal A nor the extract in skin cancer. Cyclin B1 decrease is important for the exit from mitosis and for the cytokinesis (Takizawa & Morgan, 2000, as cited in El-Najjar et al., 2007). Another protein which was differentially modulated in skin *vs* colon cancer is p53. In colon cancer, the crude extract increased the expression levels of the p53

In leukemic cells, similarly to what was shown with the other two types of cancers, the extract induced pre-G1 cell cycle arrest, increased the levels of p53 and p21 as well as the ratio of Bax to Bcl-2, and decreased cyclin D1 levels. In addition, the secretion of the vascular endothelial growth factor (VEGF) was significantly decreased by Sal A and the crude extract, adding VEGF to the list of targets of Sal A (El-Sabban, unpublished findings). Finally, Sal A was tested in combination with TNP against human colon cancer cell lines (Gali-Muhtasib, unpublished findings). At low concentrations, Sal A and TNP induced G2/M cell cycle arrest when tested separately. At the same low concentrations, the combination of Sal A and TNP synergistically inhibited tumor growth and triggered apoptosis. ROS, which increased by a factor of 25 upon combination treatment, were found to be responsible for the synergistic-induced cell death. The pretreatment of the colorectal cancer cells with the antioxidant N-acetyl-L-cysteine (NAC), which diminishes the intracellular ROS, reversed the synergistic anti-proliferative effect and protected the cells from apoptosis and therefore confirmed the implication of ROS in the induction of apoptosis. Moreover, p38 kinase, the extracellular signal regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) of the mitogen-activated protein kinases (MAPK) pathway were found to be phosphorylated and thus induced after the combination treatment. Literature strongly advocates for the MAPK pathway implication in ROS induced cell death (Zhang et al., 2003). Using specific inhibitors of both ERK (usually involved in mitogenic signals and cellular proliferation), and of p38 (associated with stress along with JNK and commonly known as the stress activated protein kinases (Lewis et al., 1998)) abolished the apoptotic synergistic effect of the combination treatment and emphasized the pathway's association to cell death. Further studies with Sal A and TNP suggested a cross-talk between ROS, JNK and Bcl-2 family in the induction of apoptosis. ROS accumulation was found to induce Bax relocalization to the mitochondrial membrane on the one hand and to decrease the ani-apoptotic Bcl-2 expression levels on the other (Gali-Muhtasib, unpublished findings). Bcl-2 decrease was also associated with a maximal JNK activation which suggested that JNK might play a role in further inactivation of Bcl-2. In accordance with our findings several studies have reported the involvement of JNK but not ERK nor p38 in the phosphorylation

Taken together these results indicate a promising chemotherapeutic effect for using Sal A

tumorigenesis (Seitz et al., 1998, as cited in Ghantous et al., 2007).

protein, while the levels were unaffected in skin cancer.

and inactivation of Bcl-2 (Srivasta et al., 1999).

alone or in combination against various cancer types.

Fig. 5. Anticancer cascade triggered by the combination of Sal A and TNP in colon cancer cells. The combination caused increase in ROS and activation of MAPK molecules, ERK, JNK and p38 leading to apoptosis. It is still unclear whether apoptosis is associated with the modulation of p21, p53, and cyclin B1 proteins or the release of cytochrome c from mitochondria.
