Cancer combination therapy of the sesquiterpenoid artesunate and the selective EGFR-tyrosine kinase inhibitor erlotinib
Thomas Efferth
PII: S0944-7113(17)30164-2
DOI: 10.1016/j.phymed.2017.11.003
Reference: PHYMED 52293
To appear in: Phytomedicine
Received date: 4 August 2017
Accepted date: 8 November 2017
Please cite this article as: Thomas Efferth , Cancer combination therapy of the sesquiterpenoid artesunate and the selective EGFR-tyrosine kinase inhibitor erlotinib, Phytomedicine (2017), doi: 10.1016/j.phymed.2017.11.003
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ABSTRACT
Background: The shift from cytotoxic to targeted chemotherapy led to improved treatment outcomes in oncology. Nevertheless, many cancer patients cannot cured from their disease because of the development of drug resistance and side effects.
Purpose: There is an ongoing quest for novel compounds, which raised not only the interest in natural products but also in novel combination therapy regimens.
Study design: In this review, we report on the inhibition epidermal growth factor receptor (EGFR) by targeted small molecules and their combination with natural products from medicinal plants.
Results: The combination of erlotinib with artesunate leads to synergistic inhibition of cell growth in isobologram analyses. Artesunate is an approved anti-malaria drug, which is also active against cancer as shown in vitro, in vivo and in preliminary clinical phase I/II trials.
Conclusion: The combination of natural products (e.g. the sesquiterpenoid artesunate) and synthetic compounds (e.g. the small molecule EGFR tyrosine kinase inhibitor erlotinib) may lead to improved clinical success rates in oncology.
Keywords: Cancer, Natural products, Small molecule inhibitors, Synergy
Introduction
Currently, there is a paradigm shift in cancer chemotherapy from classical cytotoxic drugs towards targeted drugs in order to reach more efficient tumor therapies at less side effects on normal tissues. There is a huge, still growing number of potential targets for targeted chemotherapy. Among them, growth factor receptors play crucial roles for cancer development, progression and metastasis. Therapeutic interventions to silence growth factors and their signaling cascades represent treatment strategies of utmost importance in oncology. In the past two decades, we focused on human epidermal growth factor receptors (EGFR, HER).
The EGFR signaling pathway as therapeutic target
The EGFR family consists of four members: EGFR/ERBB1/HER1, HER2/ERBB2/c-neu, HER3, and HER4. Appropriate ligands bind to these receptors and activate downstream signaling pathways, which regulate proliferation, differentiation, apoptosis, metastasis, and angiogenesis of tumors. Overexpression and/or mutations of EGFR and HER2 can be observed in many epidermal tumor types. This is causatively linked to resistance towards chemo- and radiotherapy as well as well as worse prognosis for the survival chances of patients (Scagliotti et al., 2004). Several ligands are known to bind to EGF receptors, e.g. EGF, transforming growth factor-α, β-cellulin, epiregulin, HB-EGF, AR, heregulin, and neuregulins) (Riese et al., 1995; Beerli and Hynes, 1996). After ligand binding, EGFR monomers form homo- or heterodimers with the same or another EGFR family member. Dimerization allows phosphorylation at intracellular domains of the receptors and thereby activation of the downstream signaling routes. The huge flexibility and complexity for signal transduction can be envisaged by the fact that more than 10 ligands can bind to homo- and heterodimers of four receptors (Riese et al., 1995; Beerli and Hynes, 1996; Olayioye et al., 2000). This complexity is even further enhanced by various duration times of receptor signaling, receptor internalization, and recycling as well as rates of phosphorylation and dephosphorylation (Earp et al., 1995). The tyrosine kinase activity of EGFR activates specific signal transduction cascades, e.g., Raf/Mek/Erk, PI3K/PDK1/Akt, PLCγ/PKC, MAPK, and JNK signaling pathways. Point mutations or gene amplification in EGFR genes are oncogenic and lead to constitutive EGFR activation.
Because of the tremendous importance in cancer biology, EGFR represents an exquisite molecular target for cancer therapy. Therapeutic antibodies binding to extracellular epitopes of EGFR as well as small molecules targeting the intracellular tyrosine kinase domain of the receptor have been clinically established during the past years (Oliveira et al., 2006). Gefitinib (Iressa®; Astra Zeneca, DE, USA) and erlotinib (Tarceva®; OSI-774, Genentech Inc., CA, USA) are first-generation inhibitors, which inhibit EGFR by competing with ATP for the ATP binding domain (Astsaturov et al., 2006). Despite considerable improvement in the management of EGFR-expressing tumors, drug resistance can develop due to the selection of point-mutated EGFR variants (Piotrowska and Sequist, 2015; Jochum et al., 2015). As a consequence, research continues to identify novel EGFR tyrosine kinase inhibitors. In recent years, phytochemicals increasingly came into the center of interest as resources for novel EGFR-directed treatment strategies. A plethora of investigations reported not only on natural product-based inhibitors of EGFR itself, but also on inhibitors for EGFR- associated signaling molecules such as the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. This highlights the potentials to develop novel cancer drugs derived from natural resources (Kadioglu et al., 2015).
Artemisinin-type drugs
As an example to demonstrate the utility of phytochemicals to target EGFR in cancer therapy, we focus on a sesquiterpene lactone from a plant used in traditional Chinese medicine. Artemisinin from Artemisia annua L. and its derivatives turned out as showcase examples for the efficacy of phytotherapy. In 2015, the Nobel Award for Physiology or Medicine was conferred to Youyou Tu, who isolated this compound (Efferth et al., 2015). Based on her achievements, a highly effective antimalarial treatment was developed, which saved the life of millions of patients. In addition to their antimalarial activity even against otherwise drug- resistant Plasmodia (Daddy et al., 2017), artemisinin and its derivatives are also active against other diseases such viral infections, schistosomiasis, leishmaniosis, atherosclerosis, and diabetes (Efferth et al., 2002, 2008; Jiang et al., 2016; Saeed et al., 2016; Li et al., 2017). We were among the first to describe that artemisinin derivatives reveal cytotoxicity towards cancer cell lines of many different tumor types (Efferth et al., 1996; Efferth et al., 2001; Efferth et al., 2002; Efferth et al., 2003; Efferth, 2017a; 2017b). Remarkably, artemisinin-type drugs are also active in vivo against diverse syngeneic animal tumors (Moore et al., 1995; Disbrow et al., 2005) and human xenograft tumors in nude mice (Dell’Eva et al., 2004; Efferth, 2015). Compassionate uses of artemisinin derivatives and Artemisia annua preparations of veterinary and human tumors encouraged the performance of several clinical phase I/II trials (Berger et al., 2005; Breuer and Efferth, 2014; Jansen et al., 2011; Krishna et al., 2014; Rutteman et al., 2013; Singh, 2002; Zhang et al., 2008, Michaelsen et al., 2015). A recent placebo-controlled, randomized and double-blind phase II trial in colorectal carcinoma patients indicates that an add-on therapy with artesunate provides survival advantages compared to standard treatment (Krishna et al., 2014).
Combination therapy of erlotinib and artesunate
These investigations point to the potential of artemisinin and its derivatives for cancer therapy. We explored the utility of this class of compounds for tumors with activated EGFR. For this reason, we treated glioblastoma multiforme cell lines with the EGFR tyrosine kinase inhibitor erlotinib in combination with artesunate (Efferth et al., 2004). Using isobologram analysis, we observed synergistic inhibition of cell growth was observed in U-87MG.ΔEGFR cells transduced with a deletion-mutant constitutively active EGFR gene, while additive effects were present in cells transduced with wild-type EGFR (U-87MG.WT-2N), kinase- deficient EGFR (U-87MG.DK-2N), mock vector controls (U-87MG.LUX), or non-transduced parental U-87MG cells (Fig. 1). Among 9 other non-transduced glioblastoma cell lines, synergistic effects were found in two cell lines (G-210GM, G-599GM), while ART and OSI- 774 acted in an additive manner in the other seven cell lines (G-211GM, G-750GM, G- 1163GM, G-1187GM, G-1265GM, G-1301GM, and G-1408GM). Sub-additive or antagonistic effects were not observed. Genomic gains and losses of genetic material in the non-transduced cell lines as assessed by comparative genomic hybridization were correlated with the IC50 values for artesunate and erlotinib and subsequently subjected to hierarchical cluster analysis and cluster image mapping. A genomic profile of chromosomal imbalances was detected that predicted cellular response to both drugs. In conclusion, the combination treatment of artesunate and erlotinib resulted in synergistic effects in glioblastoma cells as compared to each drug alone. This indicates that improved tumor killing may be achieved in tumors with activated EGFR and reduced activity towards EGFR-directed drugs.
It is remarkable that the combination between artesunate and erlotinib finds a chemical equivalent in antimalarial and anti-schistosomal and therapies, where mefloquine is applied (Looareesuwan et al., 1992; Xiao et al., 2011; Kerschbaumer et al., 2010). Interestingly, both erlotinib and mefloquine are chinolidin derivatives. It can be hypothesized that the combination of artemisinin-type sesquiterpene lactones and chinolidin derivatives represent promising combinations, which have the ability to improve therapeutic effects. The successful combination therapy of ART/mefloquine might, thus, be taken as a clue that the combination of ART/erlotinib might also be promising to fight EGFR-positive cancer.
To get more insight into the synergistic interaction between erlotinib and artesunate, we analyzed the role of EGFR signaling pathways for the activity of artesunate towards cancer cells (Konkimalla et al., 2009). The microarray-based mRNA expression of genes involved in EGFR signaling pathway was correlated with the 50% inhibition concentrations (IC50) of 55 tumor cell lines for artesunate. Candidate genes identified by this pharmacogenomic approach were then experimentally validated by transfecting cell lines with corresponding cDNA vectors and treating them with artesunate. We found that the EGFR downstream signaling route mediated by Ras>Raf>MEK>ERK represents an important pathway determining the sensitivity of cancer cells to artesunate. The combination of erlotinib and artesunate may lead to synergistic effects by erlotinib-mediated inhibition of EGFR phosphorylation and artesunate-mediated inhibition of the Ras>Raf>MEK>ERK downstream pathway. The combinatorial treatment approaches like these might foster the development of novel molecular targeted therapies for cancer treatment.
The effects of artesunate on EGFR signaling cannot only be demonstrated in cell lines in vitro, but also in vivo. Ma et al. (2011) found that artesunate inhibited the growth of human A549 lung xenograft tumors in nude mice. Artesunate down-regulated the expression of EGFR and another downstream signaling molecule AKT at the mRNA and protein levels in vitro and in vivo. This is another hint that artesunate may not only be an effective anti-cancer drug, but that it may also enhance the effectiveness of other EGFR-directed anticancer drugs.
Zhang et al. (2013) synthesized a novel artemisinin dimer with improved water solubility at lower pH values, which are known to occur in tumors. This derivate incorporated into nanoparticles inhibited tumor growth and downregulated protein expression of three different EGFR family members (EGFR, HER2 and HER3) in breast cancer cells.
Clinical anticancer activity of artemisinin-type drugs
In a pilot clinical phase I/II trial, we investigated the benefits of orally administered artenimol (an active metabolite of artesunate) in advanced cervix carcinoma (Jansen et al., 2011). Ten patients were treated with artenimol for 28 days. Artenimol treatment induced clinical remission with a median time for the disappearance of the symptoms being 7 days. No adverse events of grade 3 or 4 occurred. Clinical symptoms, vaginal discharge and pain disappeared after treatment for three weeks. Biopsy samples analyzed by immunohistochemistry for the expression of relevant tumor markers. Among other markers, the expression of EGFR decreased during treatment. This current pilot study indicated an improvement of clinical symptoms and good tolerability of artenimol in patients with advanced carcinoma of the cervix uteri.
In another clinical pilot study, the anticancer effect and tolerability of oral artesunate in colorectal cancer was investigated (Krishna et al., 2014). This was a single center, randomized, double-blind, placebo-controlled trial. Twenty patients (artesunate = 9, placebo = 11) completed the trial per protocol. During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group developed recurrent tumor, indicating that artesunate treatment bears the potential for improving progression free survival and overall survival of patients.
The tolerability of the artemisinin-type drugs used in all these compassionate used and clinical pilot Phase I/II trials was excellent. Significant toxicities have not been observed in the published case reports and clinical pilot Phase I/II trials in several tumor types (Berger et al., 2005; Breuer and Efferth, 2014; Jansen et al., 2011; Krishna et al., 2014; Singh, 2002; Zhang et al., 2008, Michaelsen et al., 2015). The main side effects were abdominal pain, “flu- like” symptoms, as well as reversible anemia and neutropenia. No severe grade 3/4 adverse effects were recorded. Artesunate has been investigated for their tolerability and safety in thousands of patients. Meta-analyses of clinical trials showed that artemisinin-type drugs are safe (Ribeiro and Olliaro, 1998; Adjuik et al., 2004; Efferth and Kaina, 2010).
Conclusion
The combination of artesunate as selected example for a terpenoid drug and erlotinib as selected example of a synthetic small molecule inhibitors demonstrates the potential for synergistic interactions of novel hybrid combinations. It deserves further investigation to explore further hybrid combinations not only in the experimental setting but also in clinical trials.
Conflict of interest
The author filed a patent on this topic (Efferth T and Halatsch ME. Combined treatment with artesunate and an epidermal growth factor receptor inhibitor. US-Patent, US 2006/0084675 A1. EGFR-IN-7 Filed: October 17th 2005, published: April 20th 2006).