Identification of antitumoral agents against human pancreatic cancer cells from Asteraceae and Lamiaceae plant extracts

As reported by the European Society for Medical Oncology (ESMO), pancreatic cancer is the seventh most common cancer in Europe, accounting for 2.8% of cancer in men and 3.2% in women. It is the fifth leading cause cancer-related death with 70,000 estimated deaths each year [1]. Depending on the tumor stage and if surgery is possible, current treatments are based on the use of the antimetabolite 5-fluorouracil (5-FU), but also gemcitabine, nab-paclitaxel, or in combination with acid folic, such as Folfirinox (folic acid+ 5-FU or irinotecan or oxaliplatin) [2]. A reduced percentage of patients respond to these antitumor agents due to the advanced stage of the tumor when it is diagnosticated and due to the appearance of resistances. In this scenario, plants are an important source to obtain new compounds that could be used as chemotherapeutic drugs.

In the last decades, plant-derived compounds have been clinically used as anti-cancer agents [3, 4], as they demonstrate the ability to modulate several molecular pathways involved in tumor development and progression. In this sense, the expected intervention for a plant-derived extract as antitumoral agent should exert a cytotoxic effect in tumor cells, without affecting cell viability of normal cells.

In a recent work, we produced and chemically characterized several extracts obtained from Lamiaceae (Melissa officinalis or Balm and Origanum majorana or Marjoram) and Asteraceae (Achillea millefolium or Yarrow, and Calendula officinalis or Marigold) plant families. For this purpose, we produced two different extracts from each plant by applying two sequential extraction steps: first Supercritical Fluid Extraction (SFE) and then, the remained vegetal raw material was re-extracted by Ultrasonic Assisted Extraction (UAE) [5].

Herein, in the present work, we investigated the antitumoral activity of those extracts obtained from Balm, Marjoram, Yarrow and Marigold SFE and UAE extracts in pancreatic cancer cell lines models (MIA PaCa-2 and PANC-1), and we have compared their biological activity and efficiency between the two extraction approaches (Fig. 1). Furthermore, additional UAE extracts were produced using the original vegetal matrix (OVM) for comparison with those produced previously [4] using the SFE residual vegetal matrix (RVM).

Although previous works have been described the antitumoral properties exerted by Marjoram [6, 7], Balm [8, 9], Yarrow [10, 11], and Marigold [12, 13], to our knowledge, the novelty of the present work lies in the analysis of extracts produced by environmentally friendly extraction techniques together with advanced biological methods in order to obtain, test and characterize natural extracts that could be potentially used for cancer therapies. After identifying the most efficient extracts inducing pancreatic cells toxicity, we further studied the mechanisms through which they induce cell death (apoptosis or necrosis) and their potentiality to inhibit cell malignancy and invasion. We also have evaluated the putative synergism with 5-FU to propose an efficient antitumoral product as a potential adjuvant in pancreatic cancer treatment. Our findings indicate that the SFE extracts obtained from Yarrow and Marigold meet the requirements to be proposed as a promising antitumoral approach.

In this work, we have evaluated the antitumoral properties of four plants (Yarrow, Marigold, Balm and Marjoram) derived extracts in pancreatic cancer cell lines.

Importantly, two different green technologies have been used to obtain the bioactive components from these plants: Supercritical Fluid Extraction (SFE) and Ultrasonic Assisted Extraction (UAE). For UAE extractions, two different raw materials were compared -the Residual Vegetable Material obtained after the SFE step (RVM), and the Original Vegetable Material (OVM)-.

Yarrow and Marigold extracts obtained by SFE were the most effective ones on inhibiting cell proliferation, exhibiting similar values of IC50, TGI and LC50 in MIA PaCa-2 cells (Table 1). These results are within the range of those previously reported for Marigold extract obtained by Laser extraction in pancreatic cancer, leukemia and fibrosarcoma (IC50 = 60 μg/mL) [12]; or by those obtained by ethyl alcohol maceration in melanoma (IC50 = 50 μg/mL) [17].

Yarrow extracts obtained by UAE (Ethanol) from OVM and RVM were also effective on inhibiting cell viability. The concentration needed to achieve 50% of growth inhibition (GI50 = 49,4 ± 25,7 μg/mL) is within the interval of that obtained in other works by methanolic stirring extraction in hepatocellular (39.02 ± 2.9 μg/mL) and cervical carcinomas (47.1 ± 1.8 μg/mL) models [18].

Conversely and related to Lamiaceae family plants (Balm and Marjoram), none of the extracts (nor SFE, nor UAE) had any effect on the range of concentration tested (Table 1, Fig. 2). Although it has been described ethanolic Marjoram extract to promote lymphoblastic leukemia cell death [19], and Balm extract in hepatocellular and gastric carcinomas [20], in both cases the range of concentrations were around one thousand times higher than the ones tested in this work (IC50 = 8 mg/mL for Marjoram and around 70 mg/mL for Balm).

Thus, Marigold and Yarrow SFE extracts displayed better dose-dependence activity compared to the UAE and other extraction methodologies, both in MIA PaCa-2 and in the more resistant model, PANC-1. Indeed, here, Yarrow and Marigold SFE extracts have a IC50 dose much higher in a colon non-cancer cell line (CCD18), comparing the IC50 in a colon cancer cell line (SW-620) (Additional file 1: Figure S1, Additional file 1: Table S1), which supports a therapeutic window for the use of the extracts in combination with current therapy.

In the other hand, current treatment in clinic is mostly based on 5-fluorouracil (5-FU) and gemcitabine, even combined with other drugs. In this sense, we wanted to determine a possible synergism of Yarrow and Marigold SFE extracts with 5-FU. Thus, when pre-conditioning MIA PaCa-2 cells with Yarrow or Marigold, the cytotoxic effect mediated by the subsequent 5-FU exposure has been increasing (Fig. 7). This enhancement in 5-FU effect has been previously described for other SFE extracts, such as Rosemary in colon cancer model [21], but Yarrow and Marigold are here described for the first time. In this sense, this synergism could be considered a promise as a co-adjuvant treatment, improving patient survival, given that both extracts potentiate the effect of a first-line chemotherapeutic agent. Until date, plants extracts, as a grouped set of molecules, have been tested and used to reduce chemotherapy side effects and for improve patient’s quality of life [22], with few approaches proposing their use as a second-line therapy [23]. Thus, there is a need to promote clinical trials with plant extracts, as an ensemble of phytochemicals, after validating the antiproliferative hypothesis in murine models.

Further describing the effect of Yarrow and Marigold SFE extracts, and regarding their mechanism of action, we have found that both extracts induce apoptosis through caspase 3/7 cleavage (Fig. 4), although Marigold has also shown to promote the accumulation of high levels of a necrotic cell population. These results suggest that different signaling pathways may be implicated in the induction of cell death for each extract. In this context, restoring apoptotic signaling pathways has been proposed as a strategy for cancer treatment. In fact, most of the antitumoral agents exert their effect through induction of apoptosis [24, 25]. Particularly, these results are in accordance with those described previously for Marigold [12] related with programmed cell death. Concerning Yarrow, there are few studies which describe its involvement in the induction of apoptosis, contributing this study to the first evidences of them.

Finally, we also have demonstrated that Yarrow and Marigold SFE inhibit transforming activity (Fig. 7). Growing and giving rise to 3D colonies is a way to monitor cell malignancy and the ability of stem and progenitor cells to sustain the tumor. If the proposed extracts affect the colony formation, cells are not able to evade signals that restraint their growth. In this sense, herein, we have seen that Yarrow induces a dose-dependent decrease in cell transformation and brakes the 3D sphere growth, while, Marigold induces disruption of the integrity of 3D colonies that may be due to the loose of their mesenchymal phenotype associated with an increased motility.

These results also suggest differences in the specific antitumoral mechanism of both extracts. They demonstrate that even both plants are from the same family (Asteraceae), there are differences in the way they exert their bioactive effect against pancreatic tumor cells. These differences encourage further studies to better understand the molecular action of the extracts, conducting individual approaches for each plant type. Once we a deeper understanding of their anticancer profile reached, the extracts could be registered for this purpose, beyond their current use as herbal preparation in most of the European Union countries.

This work has demonstrated that Yarrow and Marigold supercritical fluid extracts (SFE), besides avoiding chemical agents for their obtaining, allow to get effective amounts of antitumor phytochemical agents with effects not only in cell viability and 3D grown, but also in sensitizing tumor cells to chemotherapeutic agents such as 5-FU.

Related to Marjoram and Balm, their derived extracts have shown no cytotoxic effect in the pancreatic cell lines tested.