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Appl Biochem Biotechnol (2017) 181:1372–1387

DOI 10.1007/s12010-016-2290-6

In Vitro and in Vivo Anticancer Activity of Sophorolipids


to Human Cervical Cancer

Hui Li 1 & Wei Guo 1 & Xiao-jing Ma 1 & Jia-shan Li 1 &


Xin Song 1,2

Received: 2 June 2016 / Accepted: 10 October 2016 /


Published online: 29 October 2016
# Springer Science+Business Media New York 2016

Abstract Six characteristic di-acetylated lactonic sophorolipids with C16:1, C16:0, C18:0, C18:1,
C18:2, and C18:3 fatty acid were obtained from Starmerella bombicola CGMCC 1576. In order to
confirm their anticancer activity against human cervical cancer cells and reveal the structure-activity
relationships, their anti-proliferation effects on HeLa and CaSki cells were estimated. The cytotoxicity
of sophorolipid molecules with different degrees of unsaturation was proved to be influenced by
carbon chain length of sophorolipids. The longer the carbon chain length, the stronger the cytotoxicity
of sophorolipids. The inhibitory mechanism of a di-acetylated lactonic C18:1 sophorolipid on HeLa
cells was investigated. The cells developed many features of apoptosis and cell cycle was blocked at
G0 phase and partly at G2 phase. The expression of CHOP and Bip/GRP78 was induced. Caspase-12
and caspase-3 were both activated. However, mitochondrial membrane potential and concentration of
cytosolic cytochrome C did not change. The induced apoptosis of HeLa cells was probably triggered
through endoplasmic reticulum signaling pathway without involvement of mitochondria. In vivo, 5,
50, and 500 mg/kg lactonic sophorolipids showed 29.90, 41.24, and 52.06 % of inhibition without
significant toxicity to tumor-bearing mice, respectively. Our findings may suggest a potential use of
sophorolipids in human cervical cancer treatment.

Keywords Sophorolipids . Anticancer activity . Human cervical cancer . In vivo . In vitro

Abbreviations
SLs Sophorolipids
C18:3 DLSL Di-acetylated lactonic SL with a C18:3 fatty acid

Electronic supplementary material The online version of this article (doi:10.1007/s12010-016-2290-6)


contains supplementary material, which is available to authorized users.

* Xin Song
songx@sdu.edu.cn

1
State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan,
Shandong 250100, China
2
National Glycoengineering Research Centre, Shandong University, Shan Da Nan Road 27, Jinan,
Shandong 250100, China
Appl Biochem Biotechnol (2017) 181:1372–1387 1373

C18:2 DLSL Di-acetylated lactonic SL with a C18:2 fatty acid


C18:1 DLSL Di-acetylated lactonic SL with a C18:1 fatty acid
C18:0 DLSL Di-acetylated lactonic SL with a C18:0 fatty acid
C16:1 DLSL Di-acetylated lactonic SL with a C16:1 fatty acid
C16:0 DLSL Di-acetylated lactonic SL with a C16:0 fatty acid
HPLC High-performance liquid chromatography
MTT 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenytetrazolium
TEM Transmission electron microscopy
MMP Mitochondrial membrane potential
ER Endoplasmic reticulum

Introduction

Human cervical cancer is one of the most common malignancies among females in terms
of incidence and mortality rates. As a disease with alarming prevalence, over 500,000
new cervical cancer cases were diagnosed annually with mortality rate of 270,000
worldwide [1]. The commonly used therapeutic methods for cervical cancer, such as
radical hysterectomy, radiotherapy, and chemotherapy, usually will bring serious side
effects to the patients and great economic burden to their families. In addition, drug
resistance is also a big difficulty in cancer treatment. Therefore, development of effica-
cious and less toxic anti-cervical cancer drugs will be of great importance in the clinic
treatment of cervical cancer.
Sophorolipids (SLs), an important microbial extracellular glycolipid biosurfactant, are
attracting great interest in pharmaceutical area as potential anticancer medicines because of
their varied biological activities. SLs were shown to have anticancer activities against
certain types of cancer cells such as human promyelocytic leukemia cell line HL60 [2],
human pancreatic cancer cells [3], human hepatoma cell line H7402, human lung adeno-
carcinoma cell line A549 and human chronic myelogenous leukemia cell line K562 [4],
glioma cell line LN-229 [5], human esophageal cancer cell KYSE 109 and KYSE 450 [6],
and MDA-MB-231 breast cancer cells [7]. However, the inhibitory effects of SL molecules
with specific structures on other cancer cells especially on human cervical cancer cells were
not very clear.
SLs bear a sophorose as a hydrophilic head and a hydroxy fatty acid as a hydrophobic tail.
Natural SLs obtained from the fermentation broth of yeasts are a mixture of slightly different
SL molecules varying in sophorose moiety (acetyl group position and acetylation degree), fatty
acid moiety (hydroxy group position, unsaturation degree and chain length) and lactonization
or not. Typically, the fatty acid tail of SLs is limited to 16 or 18 carbon atoms [8, 9]. It has been
proved that SL structures (acetylation degree of sophorose, unsaturation degree of hydroxy
fatty acid, and lactonization or not) could all affect their cytotoxicity [6, 7]. However, to our
knowledge, the influence of carbon chain length of fatty acid moiety on their anticancer
activity has so far not been studied in detail.
Different anticancer mechanisms for SLs have been proposed. Both Isoda et al. [2] and
Joshi-Navare et al. [5] found that SLs might induce differentiation of human promyelocytic
leukemia cell line HL60 and glioma cell line LN-229 while Chen et al. [10] demonstrated
that a di-acetylated lactonic C18:1 SL molecule (C18:1 DLSL) induced apoptosis of human
liver cancer cells H7402. However, the signaling pathway of apoptosis induced by C18:1
1374 Appl Biochem Biotechnol (2017) 181:1372–1387

DLSL was not revealed. Besides, although it is well accepted that SLs have anticancer
activity in vitro, whether they have anti-tumor activity in vivo has not been reported before.
In present work, we try to confirm the cytotoxicity of SLs to human cervical cancer cells
and reveal the effects of degree of unsaturation and chain length of the hydroxy fatty acid part
in SL molecules on their cytotoxicity. Furthermore, the mechanism of C18:1 DLSL-induced
apoptosis especially the signaling pathway was investigated in vitro. We also primarily studied
the anti-cervical cancer activity of SLs in vivo for the first time.

Material and Methods

Production, Purification, and Elucidation of SL Molecules

SLs were synthesized by fermentation of Starmerella bombicola CGMCC 1576 [11] which was
originally identified as Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 [4]. The
strain was activated in a 300-ml flask containing 50 ml YEPD (the medium compositions (g/l):
glucose 20.0, peptone 20.0 and yeast extract 10.0) liquid media for 24 h at 200 rpm, 30 °C. Then
the activated cells were transferred to 50 ml fermentation media containing rape seed oil or
linseed oil as hydrophobic carbon source (the fermentation medium compositions (g/l): glucose
80.0, yeast extract 3.0, KH2PO4 1.0, Na2HPO4·12H2O 1.0, MgSO4·7H2O 0.5, and rape seed oil
or linseed oil 60.0) in a 300 ml flask and incubated for 168 h at 200 rpm, 30 °C. Two volumes of
ethyl acetate were added to the fermentation broth, and the mixture was treated according to the
method described in our previous work [6] to obtain the lactonic SLs samples. Different SL
molecules in lactonic SLs samples were purified by high-performance liquid chromatography
(HPLC) [12, 13]. The structures of purified SL molecules were elucidated by positive and
negative MS and MS/MS methods on Agilent Q-TOF6510 (Agilent Technologies Inc., USA),
and the structures of the fatty acid moiety of SLs were identified by GC-MS method [13].

Cell Lines and Cell Culture

Human cervical cancer HeLa cells and CaSki cells were cultured in DMEM medium (high
glucose) (Hyclone, Thermo Scientific, Waltham, USA) and RPMI-1640 medium (Hyclone,
Thermo Scientific, Waltham, USA), both supplemented with 10 % fetal bovine serum (Gibco,
Uruguay), 100 μg/ml penicillin and 100 μg/ml streptomycin (Amersco, Solon, OH, USA), at
37 °C in a humid atmosphere with 5 % CO2 and 95 % air, respectively. Human umbilical vein
endothelial cells (HUVECs) [14] were chosen as the normal cell control and cultured in
medium 199 (M199) (Hyclone, Thermo Scientific, Waltham, USA) supplemented with 10 %
FBS, 70 mg/ml bFGF, 100 μg/ml penicillin and 100 μg/ml streptomycin on gelatin-coated
plastic dishes at 37 °C with a humidified atmosphere of 5 % CO2 in air.

MTT Assay of Cell Viability

Cell proliferation rate was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-


diphenytetrazolium; Sigma, USA) assay after the cells were treated with different SL samples
at a final concentration ranged from 10 to 80 μg/ml for 24 h (See [6]). There were 6 repeats for
each sample. The data are representatives of three independent experiments and reported as
mean ± SD; Statistical analysis was done by t test.
Appl Biochem Biotechnol (2017) 181:1372–1387 1375

Transmission Electron Microscopy (TEM) Analysis

TEM was applied to observe the intracellular ultrastructural changes. Cells were treated the
same way as our previous work [10] and examined by a JEM-1200EX electron microscope
(JEOL, Japan) at last.

TUNEL Assay

The characteristic DNA fragmentation in HeLa cells treated with or without 40 μg/ml of C18:1
DLSL for 24 h was detected using in situ cell apoptosis detection kit (DeadEnd colorimetric
TUNEL system, Promega, Promega Co., USA) according to the manufacturer’s instructions.

Flow Cytometry Assay

The cells treated with 0, 20, 30, and 40 μg/ml of C18:1 DLSL for 24 h were detached with
trypsin and collected by centrifugation at 200 g for 6 min, fixed with 70 % cold ethanol at 4 °C
for about 18 h, centrifuged at 200 g for 5 min, washed twice with PBS, and incubated in PI/
Triton X-100 staining solution with RNase A at 37 °C for 30 min in dark. DNA content of
cells was examined by Cytomics FC500 flow cytometer (Beckman Coulter, Fullerton, CA,
USA). Cell cycle distribution was analyzed by Modifit-3 program [15].

Caspase-12 and Caspase-3 Activity Assay

Caspase-12 activity was assayed by caspase-12 colorimetric assay kit (Genmed Scientifics
Inc., DE, USA). The substrate Ac-ATAD-pNA (acetyl-Ala-Thr-Ala-Asp-p-nithoaniline) could
be hydrolyzed by caspase-12, and the contents of the released p-nithoaniline were measured at
OD405. The caspase-12 activity was determined according to the instructions.
The activity of caspase-3 was ascertained by measuring the DEVD-pNA cleavage activity
using Caspase-Glo® 3/7 Assay (Promega, USA) according to the manual.

Quantitative Real-Time RT-PCR Analysis (qRT-PCR)

RNA was extracted from cell layers using RNAiso Plus (TaKaRa) after they were treated
with 0, 10, 20, 30, and 40 μg/ml C18:1 DLSL. cDNA were synthesized using
PrimeScript® RT reagent Kit With gDNA Eraser (Perfect Real Time) (TaKaRa) according
to the manufacturer’s instructions. qRT-PCR reactions were performed using SYBR®
Premix Ex Taq II kit (TaKaRa) and monitored using a LightCycler instrument (Roche,
Mannheim, Germany). The β-actin gene was selected as a reference gene. All primers were
synthesized by Beijing Dingguo Changsheng Biotechnology Co. Ltd., and the primers were
listed as follows:

CHOP-RT-For: TACCTATGTTTCACCTCCTGG,
CHOP-RT-Rev.: GAATCTGGAGAGTGAGGGCT,
GRP78-RT-For: CCAACTGTTACAATCAAGGTCTAT,
GRP78-RT-Rev.: CACGAGGAGCAGGAGGAAT,
β-actin-RT-For: CGTGGACATCCGCAAAGA,
β-actin-RT-Rev.: TCTTCATTGTGCTGGGTGC.
1376 Appl Biochem Biotechnol (2017) 181:1372–1387

PCR amplification was performed in a total volume of 20.0 μl containing 7.4 μl water,
10 μl SYBR® Premix ExTaq™, 0.8 μl of each primer (10 μM) and 1 μl cDNA template. The
thermocycler parameters consisted of an initial denaturation at 95 °C for 2 min and 40 cycles
of 95 °C 15 s, 58 °C 10 s, 72 °C 15 s, and 85 °C 5 s. After the amplification, to confirm that
only the specific product was amplified, a melting curve was generated with a temperature
gradient of 0.1 °C s−1 from 65 to 95 °C. The quantitative analysis was carried out by
LightCycler analysis software Version 4.0 (Roche, Mannheim, Germany). The standard curve
of each gene was prepared according to the method reported by Lee et al. [16]. The copy
number of each gene obtained from qRT-PCR was calculated according to its corresponding
standard curve. Each sample was assessed at least in three duplicates.

ELISA Assay for Quantitative Measurement of GRP78 and Cytochrome C (Cyt. C)

The quantitative determination of GRP78 and Cyt.C in cytoplasm of cells treated with 0, 10,
20, 30, and 40 μg/ml C18:1 DLSL was performed by ELISA method using Human immu-
noglobulin binding protein (Bip) ELISA Kit and Human Cytochrome-C ELISA Kit (Cusabio
Biotech Co., LTD), according to the instructions.

Detection of Mitochondrial Membrane Potential (MMP) Changes

For the detection of MMP changes, cells treated with 30 μg/ml C18:1 DLSL for 0, 0.5, 2, 4,
18, and 24 h were incubated with rhodamine 123 (Rh123) (5 μg/ml of final concentration) for
30 min in dark at 37 °C, harvested and suspended in PBS, respectively. The fluorescence
intensity changes were observed using a fluorescence microscope (Nikon, Japan).

Mice Tumor Xenografts

Female BALB/c nude mice, purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai,
China), approximately 60∼8 weeks old, were housed in stainless steel cages, supplied with sterile
food and autoclaved water. All procedures were performed according to the protocols approved by
the Institutional Animal Care Committee. HeLa cells (3 × 106) were administered to the right flank
region of each mouse via subcutaneous injection [17]. Seven days later, 40 tumor-bearing mice
were randomly divided into four groups (n = 10 per group): three groups were treated with SLs at 5,
50, and 500 mg/kg and the control group received vehicle (80 % ethanol). Drugs were administered
intragastrically once a day and lasted for 12 days. The weight of mice was measured every 7 days
from the day when they were administered intragastrically. Fourteen days later, mice were
sacrificed and the developed tumors were obtained and evaluated in terms of weight. Following
the analysis of tumor mass, tumor samples treated with vehicle or 50 mg/kg of SLs were chosen
randomly and prepared as frozen tissue sections for TUNEL assay.

Results and Discussion

Purification and Elucidation of SL Molecules

Rape seed oil and linseed oil were respectively employed as hydrophobic carbon sources to
expect that the different fatty acids in the two oils were converted into different fatty acid moieties
Appl Biochem Biotechnol (2017) 181:1372–1387 1377

in SLs. As shown in Fig. 1a, b, the compositions of SLs produced by S. bombicola grown on
different hydrophobic carbon sources were a little different. Some of the main molecules were
collected and their structures were elucidated by MS, MS/MS (Supplementary Table 1 and
Table 2) and GC-MS as described in previous work [13, 18]. Six characteristic di-acetylated
lactonic sophorolipids with C16:1, C16:0, C18:0, C18:1, C18:2, and C18:3 fatty acid (C16:1
DLSL, C16:0 DLSL, C18:0 DLSL, C18:1 DLSL, C18:2 DLSL, and C18:3 DLSL) were obtained
and their chemical structures were shown in Fig. 1c. The cytotoxicity of C16:1 DLSL and C16:0
DLSL has not been reported. Additionally, the cytotoxicity of the other four SLs to cervical
cancer cells was not investigated, although their cytotoxicity to human esophagealcancer cells [6]
or MDA-MB-231 breast cancer cells [7] has been reported before. Two cervical carcinoma cell
lines HeLa and CaSki cells, infected with HPV-18 and HPV-16, respectively, are frequently
utilized systems to screen drugs for cervical cancer therapy. Therefore, the anti-proliferation
effects of these SL molecules on HeLa and CaSki cells were estimated to reveal the relationship
between the structures of SL molecules and their cytotoxicity.

The Relationship Between the Structures of SL Molecules and their Cytotoxicity


to Human Cervical Cancer Cells

Our previous work [6] found that, when di-acetylated lactonic SL had 18C in fatty acid moiety,
degree of unsaturation of SLs affected their cytotoxicity to esophageal cancer cells and the

Fig. 1 Purification of SL molecules and their structures. a and b HPLC chromatogram of different crude SLs
fermented by S. bombicola on rape seed oil and linseed oil, respectively. c Chemical structures of six different SL
molecules. C18:0 DLSL, C18:1 DLSL, C18:2 DLSL, or C18:3 DLSL is di-acetylated lactonic SL with a C18:0,
C18:1, C18:2, or C18:3 fatty acid, respectively. C16:0 DLSL and C16:1 DLSL is di-acetylated lactonic SL with a
C16:0 or C16:1 fatty acid, respectively
1378 Appl Biochem Biotechnol (2017) 181:1372–1387

inhibitory effect of C18:1 DLSL was stronger than that of C18:2 DLSL and C18:0 DLSL. To
confirm the effects of unsaturation degree of fatty acid moiety of SLs on the viability of HeLa
and CaSki cells, besides the above three different SL molecules, C18:3 DLSL was also
employed. The viability of cells treated by almost all of selected SL concentrations was
significantly different from that of the untreated cells (P < 0.01). According to the MTT
results (Fig. 2a, b), except C18:2 DLSL, the other three SL molecules showed a dose-
dependent growth inhibition in both cell lines. It can be seen from the IC50 values summarized
in Table 1 that C18:1 DLSL had stronger inhibitory effect on HeLa and CaSki cells than C18:0
DLSL than C18:3 DLSL than C18:2 DLSL. This outcome indicated that the effects of degree
of unsaturation of SLs on their cytotoxicity to HeLa and CaSki cells were complicated and
there were no rules to follow. The results were a little different from that on esophageal cancer
cells [6], which might be attributed to different cancer cell types. In the other group, the
cytotoxicity of two SL molecules with 16C fatty acid, C16:0 DLSL and C16:1 DLSL, was also
studied and the two SL molecules showed similar cytotoxicity (Fig. 2c, d). The IC50 values of
the C16:0 DLSL and C16:1 DLSL to HeLa cells (62.95 and 62.78 μg/ml, respectively) were
almost the same. Similarly, the IC50 values of the two SL molecules to CaSki cells (82.23 and

Fig. 2 Cytotoxicity of di-acetylated lactonic SLs differing in unsaturation degree (a∼d), carbon chain length
(e∼h), and the selective cytotoxicity of C18:1 DLSL to cancer cells (i) by MTT assay. C18:0 DLSL, C18:1
DLSL, C18:2 DLSL, or C18:3 DLSL is di-acetylated lactonic SL with a C18:0, C18:1, C18:2, or C18:3 fatty
acid, respectively. C16:0 DLSL and C16:1 DLSL is di-acetylated lactonic SL with a C16:0 or C16:1 fatty acid,
respectively. The data are representative of three independent experiments and reported as mean ± SD; n = 6 in all
groups
Appl Biochem Biotechnol (2017) 181:1372–1387 1379

Table 1 IC50 (μg/ml) values of different SL molecules on the proliferation of HeLa and CaSki cells

SL C16:0 DLSL C16:1 DLSL C18:0 DLSL C18:1 DLSL C18:2 DLSL C18:3 DLSL

HeLa 62.95 62.78 30.24 12.23 476.46 51.49


CaSki 82.23 82.76 29.54 25.45 1966.93 85.92

82.76 μg/ml, respectively) also showed no difference (Table 1). It suggested that the
unsaturation degree of 16C fatty acid moiety of SLs had little influence on the cytotoxicity.
So, the cytotoxicity of SL molecules not only was influenced by unsaturation degree but also
by carbon chain length of fatty acid moiety in SLs.
We then examined the influence of carbon chain length of the hydroxy fatty acid in SLs on
their cytotoxicity (Fig. 2e–h). Four SL molecules were divided into two groups: one group
contained C18:1 DLSL and C16:1 DLSL and the other group included C18:0 DLSL and C16:0
DLSL. IC50 values decreased with the elongation of carbon chain length in both groups. It might
indicate that the longer the carbon chain length of SLs, the stronger their cytotoxicity. The
increase of the lipophilicity of SL molecules may contribute to their stronger cytotoxicity.
Lipophilicity could be assessed by Log P values that were predicted by ACD Labs 12 software,
the Log P values of C16:1, C18:1, C16:0, and C18:0 DLSL were 6.71 ± 0.88, 7.83 ± 0.88,
7.20 ± 0.88, and 8.32 ± 0.88, respectively. So, with the elongation of the fatty acid chain length,
the predicted Log P values of SLs increased and the IC50 values decreased. The same trend of
lipophilicity of SLs and their cytotoxicity might demonstrate that the cytotoxicity of SLs was
probably related to their lipophilicity. Ranke et al. [19] found that there was a good correlation
between the alkyl chain length in 1, 3-dialkylimidazolium salts and their cytotoxicity to
promyelocytic leukemia rat cell line IPC-81 and the rat glioma cell line C6. The cytotoxicity
increased with the increase of alkyl chain length. Then they found that cation lipophilicity was a
dominant factor for ionic cytotoxicity [20]. Lipophilicity could affect the extent and the rate of
absorption; therefore, it is an important parameter in drug design. McKeage et al. [21] found that
IC50 values of lipophilic cations bore a parabolic dependence on drug lipophilicity. The cellular
uptake rate of more lipophilic drugs was higher and the initial uptake rate showed a parabolic
dependence on drug lipophilicity but a linear relationship to IC50 values. This might indicate that
the influence of lipophilicity on cytotoxicity was attributed to the cellular uptake rate of drugs.
Therefore, SLs with higher lipophilicity might have stronger cytotoxicity to HeLa cells.
Finally, in order to identify the cytotoxicity of SLs to normal cells, HUVECs cells were
chosen as the noncancerous control and the anti-proliferative activity of C18:1 DLSL against
HeLa, CaSki, and HUVECs cells was studied. The cell viability of HUVECs was much higher
than that of HeLa and CaSki cells (Fig. 2i). This result suggested the good selective cytotox-
icity of C18:1 DLSL molecule to cancer cell lines.
According to the results, the influence of the structures of SLs on their cytotoxicity to
HeLa and CaSki cells was very complicated; the cytotoxicity of different SLs was a result of
inter-influence of unsaturation, carbon chain length of fatty acid, and acetylation of
sophorose. As HeLa cells showed good sensitivity to SL molecules and were relatively easy
to culture; C18:1 DLSL showed the strongest cytotoxicity to HeLa cells among six different
lactonic SL molecules and its primary inhibition mechanism on human liver cancer cells
H7402 has been investigated in our previous work [10]; HeLa cells and C18:1 DLSL were
applied in the subsequent studies to further identify its inhibitory mechanism on human
cervical cancer.
1380 Appl Biochem Biotechnol (2017) 181:1372–1387

Mechanistic Study on the Cytotoxic Effect of C18:1 DLSL

C18:1 DLSL Induced Apoptosis of HeLa Cells

It has been proved that C18:1 DLSL could inhibit human liver cancer cells H7402 by inducing
cell apoptosis [10] while SLs could induce differentiation of human promyelocytic leukemia
cell line HL60 and glioma cell line LN-229 [2, 5]. To investigate the inhibition mechanism of
SL against human cervical cancer cells, HeLa cells treated by C18:1 DLSL were observed by
TEM which is called as Bgolden standard^ for the evaluation of microscopic characteristics of
cell apoptosis. Untreated cells had high nucleus plasma ratio and large nucleolus while
apoptosis characteristics appeared in C18:1 DLSL-treated cells: cell shrinkage, chromatin
condensation inside the nuclear membrane and margination to be a block or crescent shape,
increase of intracytoplasmic vacuoles, integrity of nuclear membrane, nuclear fragmentation,
and appearance of apoptotic bodies (Fig. 3a).
To further detect DNA fragmentation at the single-cell level, TUNEL assay was performed.
The cells with apoptotic nuclei were stained with a dark brown color while the untreated cells
could not be stained (Fig. 3b). The results demonstrated that endonucleases were activated
when HeLa cells were treated with C18:1 DLSL.
Cell cycle distribution was measured by flow cytometry after 24 h of treatment with C18:1
DLSL at different concentrations to further study the apoptosis properties of HeLa cells
induced by SL. The sub-G1 population showed an obvious increase as the concentration of
C18:1 DLSL increased and reached 68.32 % of the total number of cells when the C18:1
DLSL concentration was 40 μg/ml (Fig. 3c). The appearance of sub-G1 peak is a characteristic
of apoptosis. Furthermore, C18:1 DLSL treatment caused an obvious change of cell cycle
distribution of HeLa cells. An accumulation of HeLa cells in G0/G1 and G2/M phase was
observed after they were exposed to 20 or 30 μg/ml C18:1 DLSL for 24 h. When the cells
were treated with 40 μg/ml of C18:1 DLSL, the percentage of cells in G0/G1 phase increased
while the percentage of cells in G2/M phase decreased a little. The data suggested that SL

Fig. 3 Evidence of C18:1 DLSL inducing apoptosis of HeLa cells. a Nuclear fragmentation of HeLa cells
treated by 40 μg/ml C18:1 DLSL for 24 h under TEM. b Apoptotic signal in HeLa cells treated by 40 μg/ml
C18:1 DLSL for 24 h detected by TUNEL assay (×400). c Cell division cycle arrest at G0/G1 phase and partly at
G2/M phase by flow cytometry. The cells treated with C18:1 DLSL at different concentrations were analyzed by
flow cytometry after PI staining, and the cell cycle distribution was analyzed by Modifit-3 program
Appl Biochem Biotechnol (2017) 181:1372–1387 1381

blocked HeLa cells at G0 phase and partly at G2 phase, resulting in the failure of the cells to
progress through the G1/S checkpoint and partly at G2/M checkpoint. Thus, the cells were on
their way to apoptosis.

The SL-Induced Apoptosis Was Probably Triggered Through ER Signaling Pathway

Among the known apoptosis pathway, there are two main possible mechanisms of cell
apoptosis induction: death receptor pathway [22] and the mitochondrial pathway [23, 24].
Recent studies showed that apoptosis can also be induced by an endoplasmic reticulum (ER)
stress-mediated pathway without the involvement of mitochondria [25]. ER is an important
organelle because of its central role in protein biosynthesis and maintenance of intracellular
calcium homeostasis. Experimentally, pharmacological agents inducing ER stress, such as
luteolin [26], cryptotanshinone [27], pachymic acid [28], etc. could also induce apoptosis,
suggesting that ER stress could induce apoptosis.
C/EBP homologous protein (CHOP) or growth arrest and DNA damage-inducible gene 153
(GADD153) is a direct consequence of ER stress and one components of the ER stress-
mediated apoptosis pathway [29]. CHOP serves as a transcriptional factor and regulates genes
involved in either survival or death [30]. High expression of CHOP might lead to cell cycle
arrest and/or apoptosis. It was reported that the induction of CHOP by nitric oxide caused
depletion of Ca2+ in pancreatic β cells and induced apoptosis [31]. The expression of CHOP at
transcriptional level in C18:1 DLSL-treated HeLa cells was detected. CHOP mRNA was
induced by C18:1 DLSL (Fig. 4a) and reached the peak at 30 μg/ml (about 57-fold of that in
untreated HeLa cells). The induced expression of CHOP suggested that SL might cause ER
stress and lead to CHOP-mediated apoptosis.
Chen et al. (2006b) found that there was an evident elevation of cytosolic Ca2+ concentration in
the C18:1 DLSL-treated H7402 cells at 40 μg/ml for 0.5 h. The increase of cytosolic calcium
concentration might result in accumulation of unfolded proteins in ER, which leads to the induction
of chaperone proteins including GRP78/Bip [32]. The expression level of inducible Bip/GRP78,
was measured by qRT-PCR and ELISA methods. As shown in Fig. 4b, c, the amount of Bip/
GRP78 expression increased at mRNA and protein level, which indicated that Bip/GRP78 was
induced in the C18:1 DLSL-treated cells. In another respect, the elevation of cytosolic Ca2+
concentration may result in activation of μ–calpain and caspase-12 and a series of reactions
containing activation of caspase-3 and Ca/Mg-dependent endonucleases [33, 34]. As shown in
Fig. 4d, the caspase-12 activity, remarkably specific to ER stress [25], in the HeLa cells treated with
30 μg/ml C18:1 DLSL was higher than that in the untreated HeLa cells. Similarly, the caspase-3
activity in the C18:1 DLSL-treated cells was also much higher than that in the untreated cells
(Fig. 4e). This meant the activation of caspase-3 in cell apoptosis induced by C18:1 DLSL. All the
evidences showed that SL-induced apoptosis might be related to ER signaling pathway.

The Role of Mitochondria in Cell Apoptosis Induced by C18:1 DLSL

Recent studies showed that some apoptotic signals might bypass mitochondria to directly
activate caspases. To determine whether mitochondria was involved in apoptosis induced by
C18:1 DLSL, MMP change was examined by the fluorescence intensity of rhodamine 123 (Rh
123) that can selectively stain the mitochondria of live cells. Compared with untreated Hela
cells, no significant changes of fluorescence intensity were observed in C18:1 DLSL-treated
HeLa cells (Fig. 5a). Although no obvious loss of MMP was detected, the concentration of
1382 Appl Biochem Biotechnol (2017) 181:1372–1387

Fig. 4 Apoptosis induced by C18:1 DLSL might be triggered through ER-stress signaling pathway. a The
induction expression of CHOP in HeLa cells treated with 0, 10, 20, 30, and 40 μg/ml C18:1 DLSL for 24 h by
qRT-PCR analysis. b and c The induction expression of Bip/GRP78 in HeLa cells treated with 0, 10, 20, 30, and
40 μg/ml C18:1 DLSL for 24 h by qRT-PCR and ELISA assay, respectively. d Assay of caspase-12 activity in
HeLa cells treated with or without 30 μg/ml C18:1 DLSL for 12 and 16 h, respectively, using caspase-12
colorimetric assay kit. e Assay of caspase-3 activity in HeLa cells treated with or without 30 μg/ml C18:1 DLSL
for 16 h using Caspase-Glo® 3/7 Assay. The data are representative of two independent experiments

cytochrome C (Cyt.C) was still determined considering that they might be released into
cytoplasm without loss of MMP [35]. ELISA assay showed that the concentration of cytosolic
Cyt. C in C18:1 DLSL-treated HeLa cells did not increase significantly (Fig. 5b). No obvious
change of both MMP and Cyt. C might suppose that the mitochondrial pathway was not
involved in the apoptosis induced by C18:1 DLSL. Therefore, the apoptosis driven by SLs
might be triggered through ER stress-mediated pathway without involvement of mitochondria.
Appl Biochem Biotechnol (2017) 181:1372–1387 1383

Fig. 5 The role of mitochondria in apoptosis induced by C18:1 DLSL. a Mitochondrial membrane potential
(MMP) changes of HeLa cells treated with 30 μg/ml C18:1 DLSL for 0.5, 2, 4, 18, and 24 h, respectively. b The
concentration of Cyt.C in cytoplasm of HeLa cells treated 0, 10, 20, and 30 μg/ml C18:1 DLSL for 24 h,
respectively

What is particularly noteworthy is that drugs might induce apoptosis through complicated
signaling pathways. Genistein-induced apoptosis in human hepatocellular carcinomas through
interaction of ER stress and mitochondrial insult [36]. Another example is apoptosis driven by
nitric oxide. Nitric oxide-induced apoptosis in pancreatic β cells was mediated by the ER
stress pathway [31] while it might be a potential inducer of the anoikis pathway because it
reduced platelet adhesion to endothelial cells and inhibited aggregation of neutrophils [37].
Therefore, there might be other signaling pathway in SL-induced apoptosis. Glycolipid
biosurfactant from Rhodococcus ruber IEGM 231 was found to have inhibitory effect on
adhesion of human peripheral blood monocyte culture to 96-well microplates when it was
applied as a hydrophobic film [38]. Similarly, in the process treated by SLs, the detachment of
treated HeLa and CaSki cells from the plates was also observed; therefore, we suppose that
1384 Appl Biochem Biotechnol (2017) 181:1372–1387

SLs might cause disruption of cell adhesion to extracellular matrix (ECM). Loss of cell
adhesion might induce cells to undergo a particular type of apoptosis known as anoikis, a
Greek word meaning Bhomelessness^ [39]. Further experiments such as the test of integrin,
focal adhesion kinase (FAK), and extracellular signal-regulated kinase (ERK1/2) will be
performed to determine if this cell detachment was linked to anoikis pathway.

Inhibitory Effect of SLs on the Growth of Human Cervical Tumor Xenografts


in Vivo

Finally, we examined their anti-tumor activity against human cervical tumor xenografts in
mice. To reduce the cost of the potential anticancer drug, crude lactonic SLs sample with
strong cytotoxicity to HeLa cells in vitro (supplementary Fig. 1) was applied. After treatment
with SLs, no significant body weight loss (Fig. 6a) and variation in the activity of GOT, GPT,
LDH and SOD (data not shown) were detected. It suggested that SLs had no significant non-
tumor toxicity to tumor-bearing mice. For the tumor, the inhibitory effects of SLs increased as
the doses increased. The average weight of tumors treated with lactonic SLs at 5, 50, and

Fig. 6 The anti-tumor effects of lactonic SLs in nude mice in vivo. Female nude mice were xenografted by s.c.
injection of HeLa cells (3 × 106). The mice were administered intragastrically daily with either vehicle control or
lactonic SLs at 5, 50, and 500 mg/kg body weight on the seventh day after inoculation. After 12-day treatment,
the mice were sacrificed to obtain the tumors. a The effects of SLs on the body weight of mice. b Weight changes
of tumor treated with or without SLs. ** stands for P < 0.01 compared with the control group. c Determination of
apoptotic signal in tumors treated with 50 mg/kg lactonic SLs by TUNEL assay (×400)
Appl Biochem Biotechnol (2017) 181:1372–1387 1385

500 mg/kg were 1.36, 1.14, and 0.93 g, which corresponded to 29.90, 41.24, and 52.06 % of
inhibition, respectively (Fig. 6b). In addition, the size of tumors treated with SLs also
decreased compared with non-treated tumors (data not shown).
SLs triggered apoptosis of cultured cancer cells. Therefore, we analyzed whether
SLs inhibited tumor growth in vivo through inducing the occurrence of apoptosis. The
tumors from mice treated with vehicle or 50 mg/kg SLs were prepared as frozen
tissue sections and then measured by TUNEL assay. The results (Fig. 6c) showed that
the tumors in control group nearly had no color while the tumors in SLs-treated group
were stained with a dark brown color. This result suggested that lactonic SLs could
also induce apoptosis of tumor in vivo. However, whether the signaling pathway was
the same as in vitro needs to be further studied. The pharmacokinetics of SLs in vivo
should also be investigated in next study.

Conclusions

The effects of the unsaturation degree and carbon chain length of fatty acid part of SL on
human cervical cancer cells were detailedly investigated in the present work, which will be
helpful in the modification of SL molecules to obtain more novel SLs as efficacious and less
toxic anticancer drugs. Since the unsaturation degree and carbon chain length of hydroxy fatty
acid had significant influence on anticancer activity of SLs, some unconventional SLs with
stronger anticancer activity can be obtained by using unconventional hydrophobic second
carbon sources or modifying the structures of SLs with enzymatic methods. SL molecules
might inhibit the proliferation of human cervical cancer cells in vitro by inducing cell apoptosis
through ER stress-mediated pathway without the involvement of mitochondria. Lactonic SLs
also had anti-tumor activity by inducing apoptosis of tumors in vivo. Our findings may suggest
a potential use of SLs as anticancer medicines in human cervical cancer treatment because of
their efficaciousness to cancer cells, and it is also advantageous due to its low toxicity to
normal cells and low cost.

Acknowledgments This study was funded by the National Natural Science Foundation of China (No.
30970052 and No. 31270089), Natural Science Foundation of Shandong Province (ZR2009BZ002), and
National Key Technology R&D Program (2011BAC02B04). We thank Professor Yaoqin Gong for providing
HeLa cells. We also thank Professor Junying Miao for donating HUVECs cells.

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